Sequential analysis of biological samples

ABSTRACT

Methods for probing multiple targets in a biological sample are provided. The methods include the steps of providing a biological sample containing multiple targets adhered to a solid support, binding at least one fluorescent probe to one or more target present in the sample, and observing a signal from the fluorescent probe. The method further includes the steps of oxidizing the bound fluorescent probe with a solution containing an oxidizing agent that substantially inactivates the fluorescent probe, binding at least one fluorescent probe to one or more target present in the sample, and observing a signal from the fluorescent probe. The methods disclosed herein also provide for multiple iterations of binding, observing, and oxidizing for deriving information about multiple targets in a single sample. An associated kit is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/560,599, entitled “Sequential Analysis of BiologicalSamples”, filed Nov. 16, 2006, which is incorporated herein byreference.

BACKGROUND

Disclosed herein are methods for sequentially analyzing a biologicalsample to discern inter alia the presence, absence, concentration,and/or spatial distribution of multiple biological targets in abiological sample.

Various methods may be used in biology and in medicine to observedifferent targets in a biological sample. For example, analysis ofproteins in histological sections and other cytological preparations maybe performed using the techniques of histochemistry,immunohistochemistry (IHC), or immunofluorescence. Analysis of proteinsin biological samples may also be performed using solid-stateimmunoassays, for example, using the techniques of western blots.

Many of the current techniques may detect only a few targets at one time(such as, IHC or fluorescence-based western blots where number oftargets detectable is limited by the fluorescence-based detectionsystem) in a single sample. Further analysis of targets may require useof additional biological samples from the source limiting the ability todetermine relative characteristics of the targets such as the presence,absence, concentration, and/or the spatial distribution of multiplebiological targets in the biological sample. Moreover, in certaininstances, a limited amount of sample may be available for analysis orthe individual sample may require further analysis. Thus, methods,agents, and devices capable of iteratively analyzing an individualsample are needed.

BRIEF DESCRIPTION

In some embodiments, methods of detecting multiple targets in abiological sample are provided. The methods include the steps ofproviding a biological sample containing multiple targets adhered to asolid support, binding at least one fluorescent probe to one or moretarget present in the sample, and observing a signal from thefluorescent probe. The method further includes the steps of oxidizingthe bound fluorescent probe with a solution containing an oxidizingagent that substantially inactivates the fluorescent probe, binding atleast one fluorescent probe to one or more target present in the sample,and observing a signal from the fluorescent probe. The process ofbinding, observing and oxidizing may be iteratively repeated.

In some embodiments, the methods include the steps of providing abiological sample containing multiple targets adhered to a membrane,binding at least one fluorescent probe to one or more target present inthe sample, and observing a signal from the fluorescent probe. Themethod further includes the steps of oxidizing the bound fluorescentprobe with a solution containing an oxidizing agent that substantiallyinactivates the fluorescent probe, binding at least one fluorescentprobe to one or more target present in the sample, and observing asignal from the fluorescent probe. The process of binding, observing andoxidizing may be iteratively repeated.

In some embodiments, kits for detection of multiple targets in abiological sample are provided. The kits include multiple probes havinga binder coupled to a fluorescent signal generator. An oxidizing agentwhen applied to the sample substantially inactivates the fluorescentsignal generator.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the absorbance spectra of Sample 1 as a function ofwavelength, after 10 minutes and 15 minutes.

FIG. 2 shows the absorbance spectra for Samples 1, 2, and 3 as afunction of wavelength.

FIG. 3 shows the absorbance spectra of Sample 4 as a function ofwavelength, after 30 minutes and 140 minutes.

FIG. 4 shows the absorbance spectra of Sample 5 as a function ofwavelength, after 20 minutes, 60 minutes, and 210 minutes.

FIG. 5 shows the absorbance spectra of Sample 6 as a function ofwavelength, after 12 minutes and 16 minutes.

FIG. 6 shows the absorbance spectra of Sample 8 as a function ofwavelength, after 22 minutes, 70 minutes, and 210 minutes.

FIG. 7 shows the absorbance spectra of Samples 9a, 10a, and 11a as afunction of wavelength.

FIG. 8 shows the absorbance spectra of Samples 9b and 10b as a functionof wavelength.

FIG. 9 shows the absorbance spectra of Samples 12a and 12b as a functionof wavelength.

FIG. 10 shows the absorbance spectra of Samples 13, 14, and 15 as afunction of wavelength.

FIG. 11 shows the absorbance spectra of Samples 16 and 17 as a functionof wavelength.

FIG. 12 shows the micrographs (at 10× magnification) of Sample 18A(before signal modification) and Sample 18B (after signal modification).

FIG. 13 shows the micrographs (at 10× magnification) of Sample 19A(before signal modification) and Sample 19B (after signal modification).

FIG. 14 shows the micrographs of Sample 20A (before signal modification)and Sample 20B (after signal modification).

FIG. 15 shows the micrographs of Samples 21A and 21B (before signalmodification) and Sample 21C (after signal modification).

FIG. 16 shows the micrographs of Samples 22A and 22B (before signalmodification) and Sample 22C (after signal modification).

FIG. 17 shows the micrographs of Samples 23A-E.

FIG. 18 shows the micrographs of Sample 24A (before signal modification)and Sample 24b (after signal modification).

FIG. 19 shows the micrographs of Sample 25A (Cy3 and Cy5 channels),Sample 25B (Cy3 and Cy5 channels), and Samples 25C-25J.

FIG. 20 shows the micrographs of Samples 26A-H.

FIG. 21 shows the micrographs of Samples 27A-C.

FIG. 22 shows the plot of average pixel intensity of the background foreach cycle in the imaging in Example 20.

FIG. 23 shows the comparison between micrographs of Samples 28A-C and29A-C.

FIG. 24 shows the micrographs of Samples 30A-D.

FIG. 25 shows the micrographs of Samples 30C-F.

FIG. 26 shows the plot of average pixel intensities for the backgroundof each cycle for Samples 30C and 30D.

FIG. 27 shows the micrographs of Samples 31A, 31B, and 31C.

FIG. 28 shows the micrographs of Samples 32A, 32B, and 32C.

FIG. 29 shows the micrographs of Samples 33, 34, 35, and 36.

FIG. 30 shows the dot blots for Samples 37, 38, 39 and 40.

FIG. 31 shows the bar chart of relative signal intensities of dots forSamples 37, 38, 39 and 40.

FIG. 32 shows the time profile of spectra of Cy3 and Cy5.

FIG. 33 shows the Cy3 absorbance values as function of time fordifferent H₂O₂ concentrations.

FIG. 34 shows the absorbance values as function of time for differentfluorophores.

FIG. 35 shows the QD 655 absorbance values as function of time for H₂O₂.

FIG. 36 shows the absorbance spectra for fluorescein using H₂O₂.

DETAILED DESCRIPTION

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms, which are used in the following description and theappended claims.

The singular forms “a” “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. Unless otherwise indicated, allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about.” Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least each numerical parameter should atleast be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques.

DEFINITIONS

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms that are used in the following description and the claimsappended hereto.

As used herein, the term “antibody” refers to an immunoglobulin thatspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of another molecule. Theantibody may be monoclonal or polyclonal and may be prepared bytechniques that are well known in the art such as immunization of a hostand collection of sera (polyclonal), or by preparing continuous hybridcell lines and collecting the secreted protein (monoclonal), or bycloning and expressing nucleotide sequences or mutagenized versionsthereof, coding at least for the amino acid sequences required forspecific binding of natural antibodies. Antibodies may include acomplete immunoglobulin or fragment thereof, which immunoglobulinsinclude the various classes and isotypes, such as IgA, IgD, IgE, IgG1,IgG2a, IgG2b and IgG3, IgM. Functional antibody fragments may includeportions of an antibody capable of retaining binding at similar affinityto full-length antibody (for example, Fab, Fv and F(ab′)₂, or Fab′). Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments may be used where appropriate so long as bindingaffinity for a particular molecule is substantially maintained.

As used herein, the term “binder” refers to a molecule that may bind toone or more targets in the biological sample. A binder may specificallybind to a target. Suitable binders may include one or more of natural ormodified peptides, proteins (e.g., antibodies, affibodies, or aptamers),nucleic acids (e.g., polynucleotides, DNA, RNA, or aptamers);polysaccharides (e.g., lectins, sugars), lipids, enzymes, enzymesubstrates or inhibitors, ligands, receptors, antigens, or haptens. Asuitable binder may be selected depending on the sample to be analyzedand the targets available for detection. For example, a target in thesample may include a ligand and the binder may include a receptor or atarget may include a receptor and the binder may include a ligand.Similarly, a target may include an antigen and the binder may include anantibody or antibody fragment or vice versa. In some embodiments, atarget may include a nucleic acid and the binder may include acomplementary nucleic acid. In some embodiments, both the target and thebinder may include proteins capable of binding to each other.

As used herein, the term “biological sample” refers to a sample obtainedfrom a biological subject, including sample of biological tissue orfluid origin obtained in vivo or in vitro. Such samples can be, but arenot limited to, body fluid (e.g., blood, blood plasma, serum, or urine),organs, tissues, fractions, and cells isolated from mammals including,humans. Biological samples also may include sections of the biologicalsample including tissues (e.g., sectional portions of an organ ortissue). Biological samples may also include extracts from a biologicalsample, for example, an antigen from a biological fluid (e.g., blood orurine).

A biological sample may be of prokaryotic origin or eukaryotic origin(e.g., insects, protozoa, birds, fish, reptiles). In some embodiments,the biological sample is mammalian (e.g., rat, mouse, cow, dog, donkey,guinea pig, or rabbit). In certain embodiments, the biological sample isof primate origin (e.g., example, chimpanzee, or human).

As used herein, the term “control probe” refers to an agent having abinder coupled to a signal generator or a signal generator capable ofstaining directly, such that the signal generator retains at least 80percent signal after contact with a solution of an oxidizing agentemployed to inactivate the fluorescent probe. A suitable signalgenerator in a control probe is not substantially oxidized orsubstantially inactivated when contacted with the oxidizing agent.Suitable examples of signal generators may include a radioactive labelor a non-oxidizable fluorophore (e.g., DAPI)

As used herein, the term “enzyme” refers to a protein molecule that cancatalyze a chemical reaction of a substrate. In some embodiments, asuitable enzyme catalyzes a chemical reaction of the substrate to form areaction product that can bind to a receptor (e.g., phenolic groups)present in the sample or a solid support to which the sample is bound. Areceptor may be exogeneous (that is, a receptor extrinsically adhered tothe sample or the solid-support) or endogeneous (receptors presentintrinsically in the sample or the solid-support). Examples of suitableenzymes include peroxidases, oxidases, phosphatases, esterases, andglycosidases. Specific examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, β-D-galactosidase, lipase, and glucoseoxidase.

As used herein, the term “enzyme substrate” refers to a chemicalcompound that is chemically catalyzed by an enzyme to form a reactionproduct. In some embodiments, the reaction product is capable of bindingto a receptor present in the sample or a solid support to which thesample is bound. In some embodiments, enzyme substrates employed in themethods herein may include non-chromogenic or non-chemiluminescentsubstrates. A signal generator may be attached to the enzyme substrateas a label.

As used herein, the term “fluorophore” or “fluorescent signal generator”refers to a chemical compound, which when excited by exposure to aparticular wavelength of light, emits light at a different wavelength.Fluorophores may be described in terms of their emission profile, or“color.” Green fluorophores (for example Cy3, FITC, and Oregon Green)may be characterized by their emission at wavelengths generally in therange of 515-540 nanometers. Red fluorophores (for example Texas Red,Cy5, and tetramethylrhodamine) may be characterized by their emission atwavelengths generally in the range of 590-690 nanometers. Examples offluorophores include, but are not limited to,4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine,derivatives of acridine and acridine isothiocyanate,5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS),4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS), N-(4-anilino-1-naphthyl)maleimide, anthranilamide, BrilliantYellow, coumarin, coumarin derivatives, 7-amino-4-methylcoumarin (AMC,Coumarin 120), 7-amino-trifluoromethylcouluarin (Coumaran 151),cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, -,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride),eosin, derivatives of eosin such as eosin isothiocyanate, erythrosine,derivatives of erythrosine such as erythrosine B and erythrosinisothiocyanate; ethidium; fluorescein and derivatives such as5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluorescein,fluorescein isothiocyanate (FITC), QFITC (XRITC); fluorescaminederivative (fluorescent upon reaction with amines); IR144; IR1446;Malachite Green isothiocyanate; 4-methylumbelliferone; orthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red,B-phycoerythrin; o-phthaldialdehyde derivative (fluorescent uponreaction with amines); pyrene and derivatives such as pyrene, pyrenebutyrate and succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron®Brilliant Red 3B-A), rhodamine and derivatives such as6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl Rhodamine,tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acidand lathanide chelate derivatives, quantum dots, cyanines, pyreliumdyes, and squaraines.

As used herein, the term “in situ” generally refers to an eventoccurring in the original location, for example, in intact organ ortissue or in a representative segment of an organ or tissue. In someembodiments, in situ analysis of targets may be performed on cellsderived from a variety of sources, including an organism, an organ,tissue sample, or a cell culture. In situ analysis provides contextualinformation that may be lost when the target is removed from its site oforigin. Accordingly, in situ analysis of targets describes analysis oftarget-bound probe located within a whole cell or a tissue sample,whether the cell membrane is fully intact or partially intact wheretarget-bound probe remains within the cell. Furthermore, the methodsdisclosed herein may be employed to analyze targets in situ in cell ortissue samples that are fixed or unfixed.

As used herein, the term “peroxidase” refers to an enzyme class thatcatalyzes an oxidation reaction of an enzyme substrate along with anelectron donor Examples of peroxidase enzymes include horseradishperoxidase, cytochrome C peroxidase, glutathione peroxidase,microperoxidase, myeloperoxidase, lactoperoxidase, or soybeanperoxidase.

As used herein, the term “peroxidase substrate” refers to a chemicalcompound that is chemically catalyzed by peroxidase to form a reactionproduct. In some embodiments, peroxidase substrates employed in themethods herein may include non-chromogenic or non-chemiluminescentsubstrates. A fluorescent signal generator may be attached to theperoxidase substrate as a label.

As used herein, the term “probe” refers to an agent having a binder anda label, such as a signal generator or an enzyme. In some embodiments,the binder and the label (signal generator or the enzyme) are embodiedin a single entity. The binder and the label may be attached directly(e.g., via a fluorescent molecule incorporated into the binder) orindirectly (e.g., through a linker, which may include a cleavage site)and applied to the biological sample in a single step. In alternativeembodiments, the binder and the label are embodied in discrete entities(e.g., a primary antibody capable of binding a target and an enzyme or asignal generator-labeled secondary antibody capable of binding theprimary antibody). When the binder and the label (signal generator orthe enzyme) are separate entities they may be applied to a biologicalsample in a single step or multiple steps. As used herein, the term“fluorescent probe” refers to an agent having a binder coupled to afluorescent signal generator.

As used herein, the term “signal generator” refers to a molecule capableof providing a detectable signal using one or more detection techniques(e.g., spectrometry, calorimetry, spectroscopy, or visual inspection).Suitable examples of a detectable signal may include an optical signal,and electrical signal, or a radioactive signal. Examples of signalgenerators include one or more of a chromophore, a fluorophore, aRaman-active tag, or a radioactive label. As stated above, with regardto the probe, the signal generator and the binder may be present in asingle entity (e.g., a target binding protein with a fluorescent label)in some embodiments. Alternatively, the binder and the signal generatormay be discrete entities (e.g., a receptor protein and alabeled-antibody against that particular receptor protein) thatassociate with each other before or upon introduction to the sample.

As used herein, the term “solid support” refers to an article on whichtargets present in the biological sample may be immobilized andsubsequently detected by the methods disclosed herein. Targets may beimmobilized on the solid support by physical adsorption, by covalentbond formation, or by combinations thereof. A solid support may includea polymeric, a glass, or a metallic material. Examples of solid supportsinclude a membrane, a microtiter plate, a bead, a filter, a test strip,a slide, a cover slip, and a test tube.

As used herein, the term “specific binding” refers to the specificrecognition of one of two different molecules for the other compared tosubstantially less recognition of other molecules. The molecules mayhave areas on their surfaces or in cavities giving rise to specificrecognition between the two molecules arising from one or more ofelectrostatic interactions, hydrogen bonding, or hydrophobicinteractions. Specific binding examples include, but are not limited to,antibody-antigen interactions, enzyme-substrate interactions,polynucleotide interactions, and the like. In some embodiments, a bindermolecule may have an intrinsic equilibrium association constant (KA) forthe target no lower than about 10⁵ M⁻¹ under ambient conditions such asa pH of about 6 to about 8 and temperature ranging from about 0° C. toabout 37° C.

As used herein, the term “target,” refers to the component of abiological sample that may be detected when present in the biologicalsample. The target may be any substance for which there exists anaturally occurring specific binder (e.g., an antibody), or for which aspecific binder may be prepared (e.g., a small molecule binder or anaptamer). In general, a binder may bind to a target through one or morediscrete chemical moieties of the target or a three-dimensionalstructural component of the target (e.g., 3D structures resulting frompeptide folding). The target may include one or more of natural ormodified peptides, proteins (e.g., antibodies, affibodies, or aptamers),nucleic acids (e.g., polynucleotides, DNA, RNA, or aptamers);polysaccharides (e.g., lectins or sugars), lipids, enzymes, enzymesubstrates, ligands, receptors, antigens, or haptens. In someembodiments, targets may include proteins or nucleic acids.

The invention includes embodiments that relate generally to methodsapplicable in analytical, diagnostic, or prognostic applications such asanalyte detection, histochemistry, immunohistochemistry, orimmunofluorescence. In some embodiments, the methods disclosed hereinmay be particularly applicable in histochemistry, immunostaining,immunohistochemistry, immunoassays, or immunofluorescence. In someembodiments, the methods disclosed herein may be particularly applicablein immunoblotting techniques, for example, western blots or immunoassayssuch as enzyme-linked immunosorbent assays (ELISA).

The disclosed methods relate generally to detection of multiple targetsin a single biological sample. In some embodiments, methods of detectingmultiple targets in a single biological sample using the same detectionchannel are disclosed. The targets may be present on the surface ofcells in suspension, on the surface of cytology smears, on the surfaceof histological sections, on the surface of DNA microarrays, on thesurface of protein microarrays, or on the surface of solid supports(such as gels, blots, glass slides, beads, or ELISA plates).

The methods disclosed herein may allow detection of a plurality oftargets in the same biological sample with little or no effect on theintegrity of the biological sample. Detecting the targets in the samebiological sample may further provide spatial information about thetargets in the biological sample. Methods disclosed herein may also beapplicable in analytical applications where a limited amount ofbiological sample may be available for analysis and the same sample mayhave to be processed for multiple analyses. Methods disclosed herein mayalso facilitate multiple analyses of solid-state samples (e.g., tissuesections) or samples adhered to a solid support (e.g., blots) withoutsubstantially stripping the probes and the targets. Furthermore, thesame detection channel may be employed for detection of differenttargets in the sample, enabling fewer chemistry requirements foranalyses of multiple targets. The methods may further facilitateanalyses based on detection methods that may be limited in the number ofsimultaneously detectable targets because of limitations of resolvablesignals. For example, using fluorescent-based detection, the number oftargets that may be simultaneously detected may be limited to about fouras only about four fluorescent signals may be resolvable based on theirexcitation and emission wavelength properties. In some embodiments, themethods disclosed herein may allow detection of greater than fourtargets using fluorescent-based detection system.

In some embodiments, the method of detecting multiple targets in abiological sample includes sequential detection of targets in thebiological sample. The method generally includes the steps of detectinga first target in the biological sample, modifying the signal from thefirst target using a chemical agent, and detecting a second target inthe biological sample. The method may further include repeating the stepof modification of signal from the second target followed by detecting athird target in the biological sample, and so forth.

In some embodiments, the method includes the steps of contacting abiological sample with a first probe and physically binding a firstprobe to a first target. The method further includes observing a firstsignal from the first probe. A chemical agent is applied to the probe tomodify the first signal. The method further includes contacting thebiological sample with a second probe and physically binding the secondprobe to a second target in the biological sample followed by observinga second signal from the second probe.

In other embodiments, the method includes the steps of providing asample containing multiple targets and binding at least one probe havinga binder coupled to an enzyme to one or more target present in thesample. The method further includes reacting the bound probe with anenzyme substrate coupled to a fluorescent signal generator and observinga signal from the fluorescent signal generator. A solution including anoxidizing agent that substantially inactivates both the fluorescentsignal generator and the enzyme is applied to the sample. The methodfurther includes binding at least one subsequent probe having a bindercoupled to an enzyme to one or more target present in the sample. Themethod further includes reacting the bound probe with an enzymesubstrate coupled to a fluorescent signal generator and observing asignal from the fluorescent signal generator.

In yet other embodiments, the method includes the steps of providing abiological sample containing multiple targets adhered to a solid supportand binding at least one fluorescent probe to one or more target presentin the sample. The method further includes observing a signal from thebound fluorescent probe. The bound fluorescent probe is oxidized with anoxidizing agent that substantially inactivates the fluorescent probe.The method further includes binding at least one subsequent fluorescentprobe to one or more target present in the sample followed by observinga signal from the subsequent bound fluorescent probe.

In yet other embodiments, the method includes the steps of providing abiological sample containing multiple targets adhered to a solid supportand binding at least one fluorescent probe to one or more target presentin the sample. The method further includes binding at least one controlprobe to one or more target in the sample. The method further includesobserving a signal from the bound fluorescent probe and a control signalfrom the control probe. The bound fluorescent probe is oxidized with anoxidizing agent that substantially inactivates the fluorescent probe andnot the control probe. The method further includes binding at least onesubsequent fluorescent probe to one or more target present in the samplefollowed by observing a signal from the subsequent bound fluorescentprobe.

Biological Samples

A biological sample in accordance with one embodiment of the inventionmay be solid or fluid. Suitable examples of biological samples mayinclude, but are not limited to, but are not limited to, cultures,blood, plasma, serum, saliva, cerebral spinal fluid, pleural fluid,milk, lymph, sputum, semen, urine, stool, tears, saliva, needleaspirates, external sections of the skin, respiratory, intestinal, andgenitourinary tracts, tumors, organs, cell cultures or cell cultureconstituents, or solid tissue sections. In some embodiments, thebiological sample may be analyzed as is, that is, without harvest and/orisolation of the target of interest. In an alternate embodiment, harvestand isolation of targets may be performed prior to analysis. In someembodiments, the methods disclosed herein may be particularly suitablefor in-vitro analysis of biological samples.

A biological sample may include any of the aforementioned samplesregardless of their physical condition, such as, but not limited to,being frozen or stained or otherwise treated. In some embodiments, abiological sample may include compounds which are not naturallyintermixed with the sample in nature such as preservatives,anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.

In some embodiments, a biological sample may include a tissue sample, awhole cell, a cell constituent, a cytospin, or a cell smear. In someembodiments, a biological sample essentially includes a tissue sample. Atissue sample may include a collection of similar cells obtained from atissue of a biological subject that may have a similar function. In someembodiments, a tissue sample may include a collection of similar cellsobtained from a tissue of a human. Suitable examples of human tissuesinclude, but are not limited to, (1) epithelium; (2) the connectivetissues, including blood vessels, bone and cartilage; (3) muscle tissue;and (4) nerve tissue. The source of the tissue sample may be solidtissue obtained from a fresh, frozen and/or preserved organ or tissuesample or biopsy or aspirate; blood or any blood constituents; bodilyfluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid,or interstitial fluid; or cells from any time in gestation ordevelopment of the subject. In some embodiments, the tissue sample mayinclude primary or cultured cells or cell lines.

In some embodiments, a biological sample includes tissue sections fromhealthy or diseases tissue samples (e.g., tissue section from colon,breast tissue, prostate). A tissue section may include a single part orpiece of a tissue sample, for example, a thin slice of tissue or cellscut from a tissue sample. In some embodiments, multiple sections oftissue samples may be taken and subjected to analysis, provided themethods disclosed herein may be used for analysis of the same section ofthe tissue sample with respect to at least two different targets (atmorphological or molecular level). In some embodiments, the same sectionof tissue sample may be analyzed with respect to at least four differenttargets (at morphological or molecular level). In some embodiments, thesame section of tissue sample may be analyzed with respect to greaterthan four different targets (at morphological or molecular level). Insome embodiments, the same section of tissue sample may be analyzed atboth morphological and molecular levels.

A tissue section, if employed as a biological sample may have athickness in a range that is less than about 100 micrometers, in a rangethat is less than about 50 micrometers, in a range that is less thanabout 25 micrometers, or in range that is less than about 10micrometers.

In some embodiments, a biological sample or the targets in thebiological sample may be adhered to a solid support. A solid support mayinclude microarrays (e.g., DNA or RNA microarrays), gels, blots, glassslides, beads, or ELISA plates. In some embodiments, a biological sampleor the targets in the biological sample may be adhered to a membraneselected from nylon, nitrocellulose, and polyvinylidene difluoride. Insome embodiments, the solid support may include a plastic surfaceselected from polystyrene, polycarbonate, and polypropylene.

Targets

A target may be present on the surface of a biological sample (forexample, an antigen on a surface of a tissue section) or present in thebulk of the sample (for example, an antibody in a buffer solution). Insome embodiments, a target may not be inherently present on the surfaceof a biological sample and the biological sample may have to beprocessed to make the target available on the surface (e.g., antigenrecovery, enzymatic digestion, epitope retrieval, or blocking). In someembodiments, the target may be present in a body fluid such as blood,blood plasma, serum, or urine. In some other embodiments, the target maybe fixed in a tissue, either on a cell surface, or within a cell.

Suitability of targets to be analyzed may be determined by the type andnature of analysis required for the biological sample. In someembodiments, a target may provide information about the presence orabsence of an analyte in the biological sample. In another embodiment, atarget may provide information on a state of a biological sample. Forexample, if the biological sample includes a tissue sample, the methodsdisclosed herein may be used to detect targets that may help incomparing different types of cells or tissues, comparing differentdevelopmental stages, detecting the presence of a disease orabnormality, or determining the type of disease or abnormality.

Targets may include one or more of peptides, proteins (e.g., antibodies,affibodies, or aptamers), nucleic acids (e.g., polynucleotides, DNA,RNA, or aptamers); polysaccharides (e.g., lectins or sugars), lipids,enzymes, enzyme substrates, ligands, receptors, antigens, or haptens. Insome embodiments, targets may essentially include proteins or nucleicacids. One or more of the aforementioned targets may be characteristicof particular cells, while other targets may be associated with aparticular disease or condition. In some embodiments, targets that maybe detected and analyzed using the methods disclosed herein may include,but are not limited to, prognostic targets, hormone or hormone receptortargets, lymphoid targets, tumor targets, cell cycle associated targets,neural tissue and tumor targets, or cluster differentiation targets

Suitable examples of prognostic targets may include enzymatic targetssuch as galactosyl transferase II, neuron specific enolase, protonATPase-2, or acid phosphatase.

Suitable examples of hormone or hormone receptor targets may includehuman chorionic gonadotropin (HCG), adrenocorticotropic hormone,carcinoembryonic antigen (CEA), prostate-specific antigen (PSA),estrogen receptor, progesterone receptor, androgen receptor, gC1q-R/p33complement receptor, IL-2 receptor, p75 neurotrophin receptor, PTHreceptor, thyroid hormone receptor, or insulin receptor.

Suitable examples of lymphoid targets may includealpha-1-antichymotrypsin, alpha-1-antitrypsin, B cell target, bcl-2,bcl-6, B lymphocyte antigen 36 kD, BM1 (myeloid target), BM2 (myeloidtarget), galectin-3, granzyme B, HLA class I Antigen, HLA class II (DP)antigen, HLA class II (DQ) antigen, HLA class II (DR) antigen, humanneutrophil defensins, immunoglobulin A, immunoglobulin D, immunoglobulinG, immunoglobulin M, kappa light chain, kappa light chain, lambda lightchain, lymphocyte/histocyte antigen, macrophage target, muramidase(lysozyme), p80 anaplastic lymphoma kinase, plasma cell target,secretory leukocyte protease inhibitor, T cell antigen receptor (JOVI1), T cell antigen receptor (JOVI 3), terminal deoxynucleotidyltransferase, or unclustered B cell target.

Suitable examples of tumour targets may include alpha fetoprotein,apolipoprotein D, BAG-1 (RAP46 protein), CA19-9 (sialyl lewisa), CA50(carcinoma associated mucin antigen), CA125 (ovarian cancer antigen),CA242 (tumour associated mucin antigen), chromogranin A, clusterin(apolipoprotein J), epithelial membrane antigen, epithelial-relatedantigen, epithelial specific antigen, gross cystic disease fluidprotein-15, hepatocyte specific antigen, heregulin, human gastric mucin,human milk fat globule, MAGE-1, matrix metalloproteinases, melan A,melanoma target (HMB45), mesothelin, metallothionein, microphthalmiatranscription factor (MITF), Muc-1 core glycoprotein. Muc-1glycoprotein, Muc-2 glycoprotein, Muc-5AC glycoprotein, Muc-6glycoprotein, myeloperoxidase, Myf-3 (Rhabdomyosarcoma target), Myf-4(Rhabdomyosarcoma target), MyoD1 (Rhabdomyosarcoma target), myoglobin,nm23 protein, placental alkaline phosphatase, prealbumin, prostatespecific antigen, prostatic acid phosphatase, prostatic inhibin peptide,PTEN, renal cell carcinoma target, small intestinal mucinous antigen,tetranectin, thyroid transcription factor-1, tissue inhibitor of matrixmetalloproteinase 1, tissue inhibitor of matrix metalloproteinase 2,tyrosinase, tyrosinase-related protein-1, villin, or von Willebrandfactor.

Suitable examples of cell cycle associated targets may include apoptosisprotease activating factor-1, bcl-w, bcl-x, bromodeoxyuridine, CAK(cdk-activating kinase), cellular apoptosis susceptibility protein(CAS), caspase 2, caspase 8, CPP32 (caspase-3), CPP32 (caspase-3),cyclin dependent kinases, cyclin A, cyclin B1, cyclin D1, cyclin D2,cyclin D3, cyclin E, cyclin G, DNA fragmentation factor (N-terminus),Fas (CD95), Fas-associated death domain protein, Fas ligand, Fen-1,IPO-38, Mcl-1, minichromosome maintenance proteins, mismatch repairprotein (MSH2), poly (ADP-Ribose) polymerase, proliferating cell nuclearantigen, p16 protein, p27 protein, p34cdc2, p57 protein (Kip2), p105protein, Stat 1 alpha, topoisomerase I, topoisomerase II alpha,topoisomerase III alpha, or topoisomerase II beta.

Suitable examples of neural tissue and tumor targets may include alpha Bcrystallin, alpha-internexin, alpha synuclein, amyloid precursorprotein, beta amyloid, calbindin, choline acetyltransferase, excitatoryamino acid transporter 1, GAP43, glial fibrillary acidic protein,glutamate receptor 2, myelin basic protein, nerve growth factor receptor(gp75), neuroblastoma target, neurofilament 68 kD, neurofilament 160 kD,neurofilament 200 kD, neuron specific enolase, nicotinic acetylcholinereceptor alpha4, nicotinic acetylcholine receptor beta2, peripherin,protein gene product 9, S-100 protein, serotonin, SNAP-25, synapsin I,synaptophysin, tau, tryptophan hydroxylase, tyrosine hydroxylase, orubiquitin.

Suitable examples of cluster differentiation targets may include CD1a,CD1b, CD1c, CD1d, CD1e, CD2, CD3delta, CD3epsilon, CD3gamma, CD4, CD5,CD6, CD7, CD8alpha, CD8beta, CD9, CD10, CD11a, CD11b, CD11c, CDw12,CD13, CD14, CD15, CD15s, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21,CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33,CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c,CD42d, CD43, CD44, CD44R, CD45, CD46, CD47, CD48, CD49a, CD49b, CD49c,CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57,CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD65s,CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72,CD73, CD74, CDw75, CDw76, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83,CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CDw93, CD94,CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105,CD106, CD107a, CD107b, CDw108, CD109, CD114, CD115, CD116, CD117,CDw119, CD120a, CD120b, CD121a, CDw121b, CD122, CD123, CD124, CDw125,CD126, CD127, CDw128a, CDw128b, CD130, CDw131, CD132, CD134, CD135,CDw136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143,CD144, CDw145, CD146, CD147, CD148, CDw149, CDw150, CD151, CD152, CD153,CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164,CD165, CD166, and TCR-zeta.

Other suitable prognostic targets may include centromere protein-F(CENP-F), giantin, involucrin, lamin A&C (XB 10), LAP-70, mucin, nuclearpore complex proteins, p180 lamellar body protein, ran, r, cathepsin D,Ps2 protein, Her2-neu, P53, S100, epithelial target antigen (EMA), TdT,MB2, MB3, PCNA, or Ki67.

Probes

In some embodiments, a binder and a label (signal generator or anenzyme) may be coupled to each other directly (that is without anylinkers). In other embodiments, a binder and a label (signal generatoror an enzyme) may be coupled to each other via a linker. As used herein,“coupled” generally refers to two entities (for example, binder andsignal generator) stably bound to one another by any physicochemicalmeans. The nature of the coupling may be such that it does notsubstantially impair the effectiveness of either entity. A binder and alabel may be coupled to each other through covalent or non-covalentinteractions. Non-covalent interactions may include, but are not limitedto, hydrophobic interactions, ionic interactions, hydrogen-bondinteractions, high affinity interactions (such as, biotin-avidin orbiotin-streptavidin complexation), or other affinity interactions.

A linker may include a form of linking structure or sequence formed dueto the non-covalent or covalent bond formation. In some embodiments, thelinker may be chemically stable, that is, may maintain its integrity inthe presence of a chemical agent. In some embodiments, the linker may besusceptible to chemical agents that is may be capable of dissociating,cleaving, or hydrolyzing in the presence of a chemical agent. Suitableexamples of linkers may include disulfide bonds (e.g., SPDP or SMPT), pHsensitive structures/sequences, structures/sequences that may be reducedin the presence of an reducing agent, structures/sequences that may beoxidized in the presence of an oxidizing agent, or any other chemical orphysical bond that may be easily manipulated (dissociated, cleaved, orhydrolyzed) in the presence of a chemical agent.

In some embodiments, a binder and a label (signal generator or anenzyme) may be chemically linked to each other through functional groupscapable of reacting and forming a linkage under suitable conditions.Suitable examples of functional group combinations may include, but arenot limited to, amine ester and amines or anilines; acyl azide andamines or anilines; acyl halides and amines, anilines, alcohols, orphenols; acyl nitrile and alcohols or phenols; aldehyde and amines oranilines; alkyl halide and amines, anilines, alcohols, phenols orthiols; alkyl sulfonate and thiols, alcohols or phenols; anhydride andalcohols, phenols, amines or anilines; aryl halide and thiols; aziridineand thiols or thioethers; carboxylic acid and amines, anilines, alcoholsor alkyl halides; diazoalkane and carboxylic acids; epoxide and thiols;haloacetamide and thiols; halotriazin and amines, anilines or phenols;hydrazine and aldehydes or ketones; hydroxyamine and aldehydes orketones; imido ester and amines or anilines; isocyanate and amines oranilines; and isothiocyanate and amines or anilines. A functional groupin one of the aforementioned functional group pair may be present in abinder and a corresponding functional group may be present in the signalgenerator or the enzyme. For example, a binder may include a carboxylicacid and the signal generator or the enzyme may include an amine,aniline, alcohol or acyl halide, or vice versa. Conjugation between thebinder and the signal generator or the enzyme may be effected in thiscase by formation of an amide or an ester linkage.

In some embodiments, the binder may be intrinsically labeled with asignal generator (for example, if the binder is a protein, duringsynthesis using a detectably labeled amino acid) or an enzyme (forexample, if the binder is an enzyme). A binder that is intrinsicallylabeled may not require a separate signal generator or an enzyme inorder to be detected. Rather the intrinsic label may be sufficient forrendering the probe detectable. In alternate embodiments, the binder maybe labeled by binding to it a specific signal generator or an enzyme(i.e., extrinsically labeled).

In some embodiments, the binder and the label (signal generator or theenzyme) are embodied in a single entity. In alternative embodiments, thebinder and the label (signal generator or the enzyme) are embodied indiscrete entities (e.g., a primary antibody capable of binding a targetand an enzyme or a signal generator-labeled secondary antibody capableof binding the primary antibody or a hapten labeled primary antibodycapable of binding a target and an enzyme or a signal generator-labeledanti-hapten antibody capable of binding the hapten labeled primaryantibody). When the binder and the signal generator or the enzyme areseparate entities they may be applied to a biological sample in a singlestep or multiple steps. In some embodiments, the binder and the label(signal generator or the enzyme) are separate entities that arepre-attached before application to the biological sample and applied tothe biological sample in a single step. In yet other embodiments, thebinder and the label (signal generator or the enzyme) are separateentities that are applied to the biological sample independently andcombine following application.

Binders

The methods disclosed herein involve the use of binders that physicallybind to the target in a specific manner. In some embodiments, a bindermay bind to a target with sufficient specificity, that is, a binder maybind to a target with greater affinity than it does to any othermolecule. In some embodiments, the binder may bind to other molecules,but the binding may be such that the non-specific binding may be at ornear background levels. In some embodiments, the affinity of the binderfor the target of interest may be in a range that is at least 2-fold, atleast 5-fold, at least 10-fold, or more than its affinity for othermolecules. In some embodiments, binders with the greatest differentialaffinity may be employed, although they may not be those with thegreatest affinity for the target.

In some embodiments, binding between the target and the binder may beaffected by physical binding. Physical binding may include bindingeffected using non-covalent interactions. Non-covalent interactions mayinclude, but are not limited to, hydrophobic interactions, ionicinteractions, hydrogen-bond interactions, or affinity interactions (suchas, biotin-avidin or biotin-streptavidin complexation). In someembodiments, the target and the binder may have areas on their surfacesor in cavities giving rise to specific recognition between the tworesulting in physical binding. In some embodiments, a binder may bind toa biological target based on the reciprocal fit of a portion of theirmolecular shapes.

Binders and their corresponding targets may be considered as bindingpairs, of which non-limiting examples include immune-type binding-pairs,such as, antigen/antibody, antigen/antibody fragment, orhapten/anti-hapten; nonimmune-type binding-pairs, such as biotin/avidin,biotin/streptavidin, folic acid/folate binding protein, hormone/hormonereceptor, lectin/specific carbohydrate, enzyme/enzyme, enzyme/substrate,enzyme/substrate analog, enzyme/pseudo-substrate (substrate analogs thatcannot be catalyzed by the enzymatic activity), enzyme/co-factor,enzyme/modulator, enzyme/inhibitor, or vitamin B12/intrinsic factor.Other suitable examples of binding pairs may include complementarynucleic acid fragments (including DNA sequences, RNA sequences, LNAsequences, and PNA sequences); Protein A/antibody; Protein G/antibody;nucleic acid/nucleic acid binding protein; orpolynucleotide/polynucleotide binding protein.

In some embodiments, the binder may be a sequence- or structure-specificbinder, wherein the sequence or structure of a target recognized andbound by the binder may be sufficiently unique to that target.

In some embodiments, the binder may be structure-specific and mayrecognize a primary, secondary, or tertiary structure of a target. Aprimary structure of a target may include specification of its atomiccomposition and the chemical bonds connecting those atoms (includingstereochemistry), for example, the type and nature of linear arrangementof amino acids in a protein. A secondary structure of a target may referto the general three-dimensional form of segments of biomolecules, forexample, for a protein a secondary structure may refer to the folding ofthe peptide “backbone” chain into various conformations that may resultin distant amino acids being brought into proximity with each other.Suitable examples of secondary structures may include, but are notlimited to, alpha helices, beta pleated sheets, or random coils. Atertiary structure of a target may be is its overall three dimensionalstructure. A quaternary structure of a target may be the structureformed by its noncovalent interaction with one or more other targets ormacromolecules (such as protein interactions). An example of aquaternary structure may be the structure formed by the four-globinprotein subunits to make hemoglobin. A binder in accordance with theembodiments of the invention may be specific for any of theafore-mentioned structures.

An example of a structure-specific binder may include a protein-specificmolecule that may bind to a protein target. Examples of suitableprotein-specific molecules may include antibodies and antibodyfragments, nucleic acids (for example, aptamers that recognize proteintargets), or protein substrates (non-catalyzable).

In some embodiments, a target may include an antigen and a binder mayinclude an antibody. A suitable antibody may include monoclonalantibodies, polyclonal antibodies, multispecific antibodies (forexample, bispecific antibodies), or antibody fragments so long as theybind specifically to a target antigen.

In some embodiments, a biological sample may include a cell or a tissuesample and the methods disclosed herein may be employed inimmunohistochemistry (IHC). Immunochemistry may involve binding of atarget antigen to an antibody-based binder to provide information aboutthe tissues or cells (for example, diseased versus normal cells).Examples of antibodies (and the corresponding diseases/disease cells)suitable as binders for methods disclosed herein include, but are notlimited to, anti-estrogen receptor antibody (breast cancer),anti-progesterone receptor antibody (breast cancer), anti-p53 antibody(multiple cancers), anti-Her-2/neu antibody (multiple cancers),anti-EGFR antibody (epidermal growth factor, multiple cancers),anti-cathepsin D antibody (breast and other cancers), anti-Bcl-2antibody (apoptotic cells), anti-E-cadherin antibody, anti-CA125antibody (ovarian and other cancers), anti-CA15-3 antibody (breastcancer), anti-CA19-9 antibody (colon cancer), anti-c-erbB-2 antibody,anti-P-glycoprotein antibody (MDR, multi-drug resistance), anti-CEAantibody (carcinoembryonic antigen), anti-retinoblastoma protein (Rb)antibody, anti-ras oneoprotein (p21) antibody, anti-Lewis X (also calledCD15) antibody, anti-Ki-67 antibody (cellular proliferation), anti-PCNA(multiple cancers) antibody, anti-CD 3 antibody (T-cells), anti-CD4antibody (helper T cells), anti-CD5 antibody (T cells), anti-CD7antibody (thymocytes, immature T cells, NK killer cells), anti-CD8antibody (suppressor T cells), anti-CD9/p24 antibody (ALL), anti-CD10(also called CALLA) antibody (common acute lymphoblastic leukemia),anti-CD11c antibody (Monocytes, granulocytes, AML), anti-CD13 antibody(myelomonocytic cells, AML), anti-CD14 antibody (mature monocytes,granulocytes), anti-CD15 antibody (Hodgkin's disease), anti-CD19antibody (B cells), anti-CD20 antibody (B cells), anti-CD22 antibody (Bcells), anti-CD23 antibody (activated B cells, CLL), anti-CD30 antibody(activated T and B cells, Hodgkin's disease), anti-CD31 antibody(angiogenesis marker), anti-CD33 antibody (myeloid cells, AML),anti-CD34 antibody (endothelial stem cells, stromal tumors), anti-CD35antibody (dendritic cells), anti-CD38 antibody (plasma cells, activatedT, B, and myeloid cells), anti-CD 41 antibody (platelets,megakaryocytes), anti-LCA/CD45 antibody (leukocyte common antigen),anti-CD45RO antibody (helper, inducer T cells), anti-CD45RA antibody (Bcells), anti-CD39, CD100 antibody, anti-CD95/Fas antibody (apoptosis),anti-CD99 antibody (Ewings Sarcoma marker, MIC2 gene product),anti-CD106 antibody (VCAM-1; activated endothelial cells),anti-ubiquitin antibody (Alzheimer's disease), anti-CD71 (transferrinreceptor) antibody, anti-c-myc (oncoprotein and a hapten) antibody,anti-cytokeratins (transferrin receptor) antibody, anti-vimentins(endothelial cells) antibody (B and T cells), anti-HPV proteins (humanpapillomavirus) antibody, anti-kappa light chains antibody (B cell),anti-lambda light chains antibody (B cell), anti-melanosomes (HMB45)antibody (melanoma), anti-prostate specific antigen (PSA) antibody(prostate cancer), anti-S-100 antibody (melanoma, salivary, glialcells), anti-tau antigen antibody (Alzheimer's disease), anti-fibrinantibody (epithelial cells), anti-keratins antibody, anti-cytokeratinantibody (tumor), anti-alpha-catenin (cell membrane), or anti-Tn-antigenantibody (colon carcinoma, adenocarcinomas, and pancreatic cancer).

Other specific examples of suitable antibodies may include, but are notlimited to, anti proliferating cell nuclear antigen, clone pc10 (SigmaAldrich, P8825); anti smooth muscle alpha actin (SmA), clone 1A4 (Sigma,A2547); rabbit anti beta catenin (Sigma, C 2206); mouse anti pancytokeratin, clone PCK-26 (Sigma, C1801); mouse anti estrogen receptoralpha, clone 1D5 (DAKO, M 7047); beta catenin antibody, clone 15B8(Sigma, C 7738); goat anti vimentin (Sigma, V4630); cycle androgenreceptor clone AR441 (DAKO, M3562); Von Willebrand Factor 7, keratin 5,keratin 8/18, e-cadherin, Her2/neu, Estrogen receptor, p53, progesteronereceptor, beta catenin; donkey anti-mouse (Jackson Immunoresearch,715-166-150); or donkey anti rabbit (Jackson Immunoresearch,711-166-152).

In some embodiments, a binder may be sequence-specific. Asequence-specific binder may include a nucleic acid and the binder maybe capable of recognizing a particular linear arrangement of nucleotidesor derivatives thereof in the target. In some embodiments, the lineararrangement may include contiguous nucleotides or derivatives thereofthat may each bind to a corresponding complementary nucleotide in thebinder. In an alternate embodiment, the sequence may not be contiguousas there may be one, two, or more nucleotides that may not havecorresponding complementary residues on the probe. Suitable examples ofnucleic acid-based binders may include, but are not limited to, DNA orRNA oligonucleotides or polynucleotides. In some embodiments, suitablenucleic acids may include nucleic acid analogs, such as dioxygenin dCTP,biotin dcTP 7-azaguanosine, azidothymidine, inosine, or uridine.

In certain embodiments, both the binder and the target may includenucleic acids. In some embodiments, a nucleic-acid based binder may forma Watson-Crick bond with the nucleic acid target. In another embodiment,the nucleic acid binder may form a Hoogsteen bond with the nucleic acidtarget, thereby forming a triplex. A nucleic acid binder that binds byHoogsteen binding may enter the major groove of a nucleic acid targetand hybridizes with the bases located there. Suitable examples of theabove binders may include molecules that recognize and bind to the minorand major grooves of nucleic acids (for example, some forms ofantibiotics.) In certain embodiments, the nucleic acid binders may formboth Watson-Crick and Hoogsteen bonds with the nucleic acid target (forexample, bis PNA probes are capable of both Watson-Crick and Hoogsteenbinding to a nucleic acid).

The length of nucleic acid binder may also determine the specificity ofbinding. The energetic cost of a single mismatch between the binder andthe nucleic acid target may be relatively higher for shorter sequencesthan for longer ones. In some embodiments, hybridization of smallernucleic acid binders may be more specific than the hybridization oflonger nucleic acid probes, as the longer probes may be more amenable tomismatches and may continue to bind to the nucleic acid depending on theconditions. In certain embodiments, shorter binders may exhibit lowerbinding stability at a given temperature and salt concentration. Bindersthat may exhibit greater stability to bind short sequences may beemployed in this case (for examples, bis PNA). In some embodiments, thenucleic acid binder may have a length in range of from about 4nucleotides to about 12 nucleotides, from about 12 nucleotides to about25 nucleotides, from about 25 nucleotides to about 50 nucleotides, fromabout 50 nucleotides to about 100 nucleotides, from about 100nucleotides to about 250 nucleotides, from about 250 nucleotides toabout 500 nucleotides, or from about 500 nucleotides to about 1000nucleotides. In some embodiments, the nucleic acid binder may have alength in a range that is greater than about 1000 nucleotides.Notwithstanding the length of the nucleic acid binder, all thenucleotide residues of the binder may not hybridize to complementarynucleotides in the nucleic acid target. For example, the binder mayinclude 50 nucleotide residues in length, and only 25 of thosenucleotide residues may hybridize to the nucleic acid target. In someembodiments, the nucleotide residues that may hybridize may becontiguous with each other. The nucleic acid binders may be singlestranded or may include a secondary structure. In some embodiments, abiological sample may include a cell or a tissue sample and thebiological sample may be subjected to in-situ hybridization (ISH) usinga nucleic acid binder. In some embodiments, a tissue sample may besubjected to in situ hybridization in addition to immunohistochemistry(IHC) to obtain desired information from the sample.

Regardless of the type of binder and the target, the specificity ofbinding between the binder and the target may also be affected dependingon the binding conditions (for example, hybridization conditions in caseof complementary nucleic acids). Suitable binding conditions may berealized by modulation one or more of pH, temperature, or saltconcentration.

A binder may be intrinsically labeled (signal generator or enzymeattached during synthesis of binder) or extrinsically labeled (signalgenerator or enzyme attached during a later step). For example for aprotein-based binder, an intrinsically labeled binder may be prepared byemploying labeled amino acids. Similarly, an intrinsically labelednucleic acid may be synthesized using methods that incorporate signalgenerator-labeled nucleotides directly into the growing nucleic acid. Insome embodiments, a binder may be synthesized in a manner such thatsignal generators or enzymes may be incorporated at a later stage. Forexample, this latter labeling may be accomplished by chemical means bythe introduction of active amino or thiol groups into nucleic acids ofpeptide chains. In some embodiments, a binder such a protein (forexample, an antibody) or a nucleic acid (for example, a DNA) may bedirectly chemically labeled using appropriate chemistries.

In some embodiments, combinations of binders may be used that mayprovide greater specificity or in certain embodiments amplification ofthe signal. Thus, in some embodiments, a sandwich of binders may beused, where the first binder may bind to the target and serve to providefor secondary binding, where the secondary binder may or may not includea label, which may further provide for tertiary binding (if required)where the tertiary binding member may include a label.

Suitable examples of binder combinations may include primaryantibody-secondary antibody, complementary nucleic acids, or otherligand-receptor pairs (such as biotin-streptavidin). Some specificexamples of suitable binder pairs may include mouse anti-myc forrecombinant expressed proteins with c-myc epitope; mouse anti-HisG forrecombinant protein with His-Tag epitope, mouse anti-express forrecombinant protein with epitope-tag, rabbit anti-goat for goat IgGprimary molecules, complementary nucleic acid sequence for a nucleicacid; mouse anti-thio for thioredoxin fusion proteins, rabbit anti-GFPfor fusion protein, jacalin for α-D-galactose; and melibiose forcarbohydrate-binding proteins, sugars, nickel couple matrix or heparin.

In some embodiments, a combination of a primary antibody and a secondaryantibody may be used as a binder. A primary antibody may be capable ofbinding to a specific region of the target and the secondary antibodymay be capable of binding to the primary antibody. A secondary antibodymay be attached to a signal generator or an enzyme before binding to theprimary antibody or may be capable of binding to a signal generator oran enzyme at a later step. In an alternate embodiment, a primaryantibody and specific binding ligand-receptor pairs (such asbiotin-streptavidin) may be used. The primary antibody may be attachedto one member of the pair (for example biotin) and the other member (forexample streptavidin) may be labeled with a signal generator or anenzyme. The secondary antibody, avidin, streptavidin, or biotin may beeach independently labeled with a signal generator or an enzyme.

In some embodiments, the methods disclosed herein may be employed in animmunostaining procedure, and a primary antibody may be used tospecifically bind the target protein. A secondary antibody may be usedto specifically bind to the primary antibody, thereby forming a bridgebetween the primary antibody and a subsequent reagent (for example asignal generator or enzyme), if any. For example, a primary antibody maybe mouse IgG (an antibody created in mouse) and the correspondingsecondary antibody may be goat anti-mouse (antibody created in goat)having regions capable of binding to a region in mouse IgG.

In some embodiments, signal amplification may be obtained when severalsecondary antibodies may bind to epitopes on the primary antibody. In animmunostaining procedure a primary antibody may be the first antibodyused in the procedure and the secondary antibody may be the secondantibody used in the procedure. In some embodiments, a primary antibodymay be the only antibody used in an immunostaining procedure.

Signal Generators

The type of signal generator suitable for the methods disclosed hereinmay depend on a variety of factors, including the nature of the analysisbeing conducted, the type of the energy source and detector used, thetype of oxidizing agent employed, the type of binder, the type oftarget, or the mode of attachment between the binder and the signalgenerator (e.g., cleavable or non-cleavable).

A suitable signal generator may include a molecule or a compound capableof providing a detectable signal. A signal generator may provide acharacteristic signal following interaction with an energy source or acurrent. An energy source may include electromagnetic radiation sourceand a fluorescence excitation source. Electromagnetic radiation sourcemay be capable of providing electromagnetic energy of any wavelengthincluding visible, infrared and ultraviolet. Electromagnetic radiationmay be in the form of a direct light source or may be emitted by a lightemissive compound such as a donor fluorophore. A fluorescence excitationsource may be capable of making a source fluoresce or may give rise tophotonic emissions (that is, electromagnetic radiation, directedelectric field, temperature, physical contact, or mechanicaldisruption). Suitable signal generators may provide a signal capable ofbeing detected by a variety of methods including optical measurements(for example, fluorescence), electrical conductivity, or radioactivity.Suitable signal generators may be, for example, light emitting, energyaccepting, fluorescing, radioactive, or quenching.

A suitable signal generator may be sterically and chemically compatiblewith the constituents to which it is bound, for example, a binder.Additionally, a suitable signal generator may not interfere with thebinding of the binder to the target, nor may it affect the bindingspecificity of the binder. A suitable signal generator may be organic orinorganic in nature. In some embodiments, a signal generator may be of achemical, peptide or nucleic acid nature.

A suitable signal generator may be directly detectable. A directlydetectable moiety may be one that may be detected directly by itsability to emit a signal, such as for example a fluorescent label thatemits light of a particular wavelength following excitation by light ofanother lower, characteristic wavelength and/or absorb light of aparticular wavelength.

A signal generator, suitable in accordance with the methods disclosedherein may be amenable to manipulation on application of a chemicalagent. In some embodiments, a signal generator may be capable of beingchemically destroyed on exposure to an oxidizing agent. Chemicaldestruction may include complete disintegration of the signal generatoror modification of the signal-generating component of the signalgenerator. Modification of the signal-generating component may includeany chemical modification (such as addition, substitution, or removal)that may result in the modification of the signal generating properties.For example, unconjugating a conjugated signal generator may result indestruction of chromogenic properties of the signal generator.Similarly, substitution of a fluorescence-inhibiting functional group ona fluorescent signal generator may result in modification of itsfluorescent properties. In some embodiments, one or more signalgenerators substantially resistant to inactivation by a specificchemical agent may be used as a control probe in the provided methods.

In some embodiments, a signal generator may be selected from a lightemissive molecule, a radioisotope (e.g., P³² or H³, ¹⁴C, ¹²⁵I and ¹³¹I),an optical or electron density marker, a Raman-active tag, an electronspin resonance molecule (such as for example nitroxyl radicals), anelectrical charge transferring molecule (i.e., an electrical chargetransducing molecule), a semiconductor nanocrystal, a semiconductornanoparticle, a colloid gold nanocrystal, a microbead, a magnetic bead,a paramagnetic particle, or a quantum dot.

In some embodiments, a signal generator may include a light-emissivemolecule. A light emissive molecule may emit light in response toirradiation with light of a particular wavelength. Light emissivemolecules may be capable of absorbing and emitting light throughluminescence (non-thermal emission of electromagnetic radiation by amaterial upon excitation), phosphorescence (delayed luminescence as aresult of the absorption of radiation), chemiluminescence (luminescencedue to a chemical reaction), fluorescence, or polarized fluorescence.

In some embodiments, a signal generator may essentially include afluorophore. In some embodiments, a signal generator may essentiallyinclude a fluorophore attached to an antibody, for example, in animmunohistochemistry analysis. Suitable fluorophores that may beconjugated to a primary antibody include, but are not limited to,Fluorescein, Rhodamine, Texas Red, VECTOR Red, ELF (Enzyme-LabeledFluorescence), Cy2, Cy3, Cy3.5, Cy5, Cy7, Fluor X, Calcein, Calcein-AM,CRYPTOFLUOR, Orange (42 kDa), Tangerine (35 kDa), Gold (31 kDa), Red (42kDa), Crimson (40 kDa), BHMP, BHDMAP, Br-Oregon, Lucifer Yellow, Alexadye family, N-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl](NBD), BODIPY, boron dipyrromethene difluoride, Oregon Green,MITOTRACKER Red, Phycoerythrin, Phycobiliproteins BPE (240 kDa) RPE (240kDa) CPC (264 kDa) APC (104 kDa), Spectrum Blue, Spectrum Aqua, SpectrumGreen, Spectrum Gold, Spectrum Orange, Spectrum Red, Infra-Red (IR)Dyes, Cyclic GDP-Ribose (cGDPR), Calcofluor White, Lissamine,Umbelliferone, Tyrosine or Tryptophan. In some embodiments, a signalgenerator may essentially include a cyanine dye. In some embodiments, asignal generator may essentially include one or more a Cy3 dye, a Cy5dye, or a Cy7 dye.

In some embodiments, the signal generator may be part of a FRET pair.FRET pair includes two fluorophores that are capable of undergoing FRETto produce or eliminate a detectable signal when positioned in proximityto one another. Some examples of donors may include Alexa 488, Alexa546, BODIPY 493, Oyster 556, Fluor (FAM), Cy3, or TTR (Tamra). Someexamples of acceptors may include Cy5, Alexa 594, Alexa 647, or Oyster656.

As described hereinabove, one or more of the aforementioned moleculesmay be used as a signal generator. In some embodiments, one or more ofthe signal generators may not be amenable to chemical destruction and acleavable linker may be employed to associate the signal generator andthe binder. In some embodiments, one or more of the signal generatorsmay be amenable to signal destruction and the signal generator mayessentially include a molecule capable of being destroyed chemically. Insome embodiments, a signal generator may include a fluorophore capableof being destroyed chemically by an oxidizing agent. In someembodiments, a signal generator may essentially include cyanine,coumarin, BODIPY, ATTO 658, or ATTO 634, capable of being destroyedchemically by an oxidizing agent. In some embodiments, a signalgenerator may include one or more a Cy3 dye, a Cy5 dye, or a Cy7 dyecapable of being destroyed or quenched.

Enzyme and Enzyme Substrates

In some embodiments, a probe may include a binder coupled to an enzyme.In some embodiments, a suitable enzyme catalyzes a chemical reaction ofthe substrate to form a reaction product that can bind to a receptor(e.g., phenolic groups) present in the sample or a solid support towhich the sample is bound. A receptor may be exogeneous (that is, areceptor extrinsically adhered to the sample or the solid-support) orendogeneous (receptors present intrinsically in the sample or thesolid-support). Signal amplification may be effected as a single enzymemay catalyze a chemical reaction of the substrate to covalently bindmultiple signal generators near the target.

In some embodiments, a suitable enzyme may also be capable of beinginactivated by an oxidizing agent. Examples of suitable enzymes includeperoxidases, oxidases, phosphatases, esterases, and glycosidases.Specific examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-D-galactosidase, lipase, and glucose oxidase. Insome embodiments, the enzyme is a peroxidase selected from horseradishperoxidase, cytochrome C peroxidase, glutathione peroxidase,microperoxidase, myeloperoxidase, lactoperoxidase, and soybeanperoxidase.

In some embodiments, a binder and an enzyme may be embodied in a singleentity, for example a protein molecule capable of binding to a targetand also catalyzing a chemical reaction of substrate. In otherembodiments, a binder and an enzyme may be embodied in separate entitiesand may be coupled by covalent bond formation or by usingligand-receptor conjugate pairs (e.g., biotin streptavidin).

An enzyme substrate may be selected depending on the enzyme employed andthe target available for binding in the sample or on the solid support.For example, in embodiments including HRP as an enzyme, a substrate mayinclude a substituted phenol (e.g., tyramine). Reaction of HRP to thetyramine may produce an activated phenolic substrate that may bind toendogeneous receptors like electron-rich moieties (such as tyrosine ortryptophan) or phenolic groups present in the surface proteins of abiological sample. In alternate embodiments, where3-methyl-2-benzothiazolinone hydrochloride (MBTH) may be employed as asubstrate along with an HRP enzyme, exogeneous receptors likep-dimethylaminobenzaldehyde (DMAB) may be adhered to the solid supportor the biological sample before reacting with the substrate.

In some embodiments, an enzyme substrate may be dephosphorylated afterreaction with the enzyme. The dephosphorylated reaction product may becapable of binding to endogeneous or exogeneous receptors (e.g.,antibodies) in the sample or the solid-support. For example, an enzymemay include alkaline phosphatase (AP) and a substrate may include NADP,substituted phosphates (e.g., nitrophenyl phosphate), or phosphorylatedbiotin. The receptors may include NAD binding proteins, antibodies tothe dephosphorylated reaction product (e.g., anti nitro-phenol), avidin,or streptavidin accordingly.

In some embodiments, an enzyme may include β-galactosidase and asubstrate may include β-galactopyranosyl-glycoside of fluorescein orcoumarin. Receptors may include antibodies to deglycosylated moieties(e.g., anti-fluorescein or anti-coumarin). In some embodiments, multipleenzyme combinations like HRP/AP may be used as an enzyme. A substratemay include phosphorylated substituted phenol e.g., tyrosine phosphate,which may be dephosphorylated by AP before reacting with HRP to form areaction product capable of binding to phenolic groups or electron richmoieties-based receptors.

A reaction product of the enzyme substrate may further be capable ofbeing providing a detectable signal. In some embodiments, enzymesubstrates employed in the methods disclosed herein may includenon-chromogenic or non-chemiluminescent substrates, that is a reactionof the enzyme and the enzyme substrate may not itself produce adetectable signal. Enzyme substrates employed in the methods disclosedherein may include an extrinsic signal generator (e.g., a fluorophore)as a label. The signal generator and the enzyme substrate may beattached directly (e.g., an enzyme substrate with a fluorescent label)or indirectly (e.g., through ligand-receptor conjugate pair). In someembodiments, a substrate may include protected functional groups (e.g.,sulfhydryl groups). After binding of the activated substrate to thereceptors, the functional group may be deprotected and conjugation to asignal generator effected using a signal generator having a thiolreactive group (e.g., maleimide or iodoacetyl).

In some embodiments, a probe may include horseradish peroxidase and thesubstrate is selected from substituted phenols (e.g., tyramine). In someembodiments, the horseradish peroxidase causes the activated phenolicsubstrate to covalently bind to phenolic groups present in the sample ora solid support to which the sample is bound. In some embodiments, aprobe may include a binder coupled to HRP and a substrate may includetyramine-coupled to a fluorophore.

Chemical Agents

A chemical agent may include one or chemicals capable of modifying thesignal generator, the enzyme, or the cleavable linker (if present)between the signal generator and the binder or the enzyme substrate. Achemical agent may be contacted with the sample in the form of a solid,a solution, a gel, or a suspension.

In some embodiments, a chemical agent may include oxidizing agents, forexample, active oxygen species, hydroxyl radicals, singlet oxygen,hydrogen peroxide, or ozone. In some embodiments, a chemical agent mayinclude hydrogen peroxide, potassium permanganate, sodium dichromate,aqueous bromine, iodine-potassium iodide, or t-butyl hydroperoxide

One or more of the aforementioned chemical agents may be used in themethods disclosed herein depending upon the susceptibility of the signalgenerator, of the enzyme, of the binder, of the target, or of thebiological sample to the chemical agent. In some embodiments, a chemicalagent that essentially does not affect the integrity of the binder, thetarget, and the biological sample may be employed. In some embodiments,a chemical agent that does not affect the specificity of binding betweenthe binder and the target may be employed.

In some embodiments, where two or more (up to four) signal generatorsmay be employed simultaneously, a chemical agent may be capable ofselectively modifying one or more signal generators. Susceptibility ofdifferent signal generators to a chemical agent may depend, in part, tothe concentration of the signal generator, temperature, or pH. Forexample, two different fluorophores may have different susceptibility toan oxidizing agent depending upon the concentration of the oxidizingagent.

In some embodiments, a chemical agent may essentially include anoxidizing agent. In other embodiments, a chemical agent may essentiallyinclude a basic solution of an oxidizing agent. In some specificembodiments, a basic solution may have a pH in a range of from about 8to about 10. In some embodiments, a basic solution (e.g., sodiumbiocarbonate) may have pH of about 10.

A suitable oxidizing agent may be selected from peroxide, sodiumperiodate, or ozone. In some embodiments, a suitable oxidizing agent mayinclude peroxide or a peroxide source and the basic solution may includehydrogen peroxide. The concentration of hydrogen peroxide in the basicsolution may be selected to substantially oxidize the fluorescent signalgenerator in a predetermined period of time. In some embodiments, theconcentration of hydrogen peroxide in the basic solution may be selectedto substantially inactivate both the fluorescent signal generator andthe enzyme in a given period of time. The concentration of hydrogenperoxide in the basic solution may be selected such that the integrityof the sample, the targets, the binder, or the target-binder specificitymay be maintained.

In some embodiments, a basic solution may include hydrogen peroxide inan amount that is in a range of from about 0.5 volume percent to about 5volume percent, in a range of from about 1 volume percent to about 4volume percent, or in a range of from about 1.5 volume percent to about3.5 volume percent. In some specific embodiments, a basic solution mayinclude hydrogen peroxide in an amount that is in a range of about 3volume percent.

In some embodiments, the basic solution may not include reagents thatstrip the binders, the targets or both the binders and the targets fromthe sample such as reducing agents or surfactants.

Sequentially Analyzing a Biological Sample, Contacting and Binding theProbe

A biological sample may be contacted with a probe to bind the probe to atarget in the biological sample. In some embodiments, a target may notbe easily accessible for binding with the probe and a biological samplemay be further processed to facilitate the binding between the targetand the binder in the probe, for example through antigen recovery,enzymatic digestion, epitope retrieval, or blocking.

In some embodiments, a probe may be contacted with the biological samplein the form of a solution. In some embodiments, a probe may include abinder coupled to a label (fluorescent signal generator or an enzyme).The binder and the label (signal generator or enzyme) may be embodied ina single molecule and the probe solution may be applied in a singlestep. Alternatively, the binder and the label (signal generator orenzyme) may be distinct entities and the probe solution may be appliedin a single step or multiple steps. In all embodiments, a control probemay further be bonded to one or more targets in the sample.

Depending on the nature of the binder, the target, and the bindingbetween the two, sufficient contact time may be allowed. In someembodiments, an excess of probe molecules (and accordingly bindermolecules) may be employed to ensure all the targets in the biologicalsample are bound. After a sufficient time has been providing for thebinding action, the sample may be contacted with a wash solution (forexample an appropriate buffer solution) to wash away any unbound probes.Depending on the concentration and type of probes used, a biologicalsample may be subjected to a number of washing steps with the same ordifferent washing solutions being employed in each step.

In some embodiments, the biological sample may be contacted with morethan one probe in the first binding step. The plurality of probes may becapable of binding different targets in the biological sample. Forexample, a biological sample may include two targets: target1 andtarget2 and two sets of probes may be used in this instance: probe1(having binder1 capable of binding to target1) and probe2 (havingbinder2 capable of binding to target2). A plurality of probes may becontacted with the biological sample simultaneously (for example, as asingle mixture) or sequentially (for example, a probe1 may be contactedwith the biological sample, followed by washing step to remove anyunbound probe1, followed by contacting a probe2 with the biologicalsample, and so forth).

The number of probes that may be simultaneously bound to the target maydepend on the type of detection employed, that is, the spectralresolution achievable. For example, for fluorescence-based signalgenerators, four different probes (providing four spectrally resolvablefluorescent signals) may be employed in accordance with the disclosedmethods. Spectrally resolvable, in reference to a plurality offluorescent signal generators, indicates that the fluorescent emissionbands of the signal generators are sufficiently distinct, that is,sufficiently non-overlapping, such that, binders to which the respectivesignal generators are attached may be distinguished on the basis of thefluorescent signal generated by the respective signal generators usingstandard photodetection systems.

In some embodiments, a biological sample may be essentially contactedwith four or less than four probes in the first binding step. Inembodiments employing enzyme-based probes, the number of probes that maybe simultaneously bound to the target may also depend on the number ofdifferent enzymes and their corresponding substrates available.

In some embodiments, a biological sample may include a whole cell, atissue sample, or the biological sample may be adhered to a microarray,a gel, or a membrane. In some embodiments, a biological sample mayinclude a tissue sample. The tissue sample may be obtained by a varietyof procedures including, but not limited to surgical excision,aspiration or biopsy. The tissue may be fresh or frozen. In someembodiments, the tissue sample may be fixed and embedded in paraffin.The tissue sample may be fixed or otherwise preserved by conventionalmethodology; the choice of a fixative may be determined by the purposefor which the tissue is to be histologically stained or otherwiseanalyzed. The length of fixation may depend upon the size of the tissuesample and the fixative used. For example, neutral buffered formalin,Bouin's or paraformaldehyde, may be used to fix or preserve a tissuesample.

In some embodiments, the tissue sample may be first fixed and thendehydrated through an ascending series of alcohols, infiltrated andembedded with paraffin or other sectioning media so that the tissuesample may be sectioned. In an alternative embodiment, a tissue samplemay be sectioned and subsequently fixed. In some embodiments, the tissuesample may be embedded and processed in paraffin. Examples of paraffinthat may be used include, but are not limited to, Paraplast, Broloid,and Tissuemay. Once the tissue sample is embedded, the sample may besectioned by a microtome into sections that may have a thickness in arange of from about three microns to about five microns. Once sectioned,the sections may be attached to slides using adhesives. Examples ofslide adhesives may include, but are not limited to, silane, gelatin,poly-L-lysine. In embodiments, if paraffin is used as the embeddingmaterial, the tissue sections may be deparaffinized and rehydrated inwater. The tissue sections may be deparaffinized, for example, by usingorganic agents (such as, xylenes or gradually descending series ofalcohols).

In some embodiments, aside from the sample preparation proceduresdiscussed above, the tissue section may be subjected to furthertreatment prior to, during, or following immunohistochemistry. Forexample, in some embodiments, the tissue section may be subjected toepitope retrieval methods, such as, heating of the tissue sample incitrate buffer. In some embodiments, a tissue section may be optionallysubjected to a blocking step to minimize any non-specific binding.

In some embodiments, the biological sample or a portion of thebiological sample, or targets present in the biological sample may beadhered on the surface of DNA microarrays, on the surface of proteinmicroarrays, or on the surface of solid supports (such as gels, blots,glass slides, beads, or ELISA plates). In some embodiments, targetspresent in the biological sample may be adhered on the surface of solidsupports. Targets in the biological sample may be adhered on the solidsupport by physical bond formation, by covalent bond formation, or both.

In some embodiments, the targets in the biological sample may be adheredto membranes and probed sequentially using the methods disclosed herein.In some embodiments, targets in the biological sample may be processedbefore contacting the sample with the membrane. For example, embodimentsinvolving methods for probing protein targets in a tissue sample mayinclude the step of extracting the target proteins a biological sampleof tissue homogenate or an extract. Solid tissues or whole cells may befirst broken down mechanically using a blender (for larger samplevolumes), using a homogenizer (smaller volumes), or by sonication.Different cell compartments and organelles may be separated usingfiltration and centrifugation techniques. Detergents, salts, and buffersmay also be employed to encourage lysis of cells and to solubilizeproteins. Similarly, embodiments involving methods for probing nucleicacids may include the step of preparing DNA or RNA fragments, forexample using restriction endonucleases (for DNA).

In some embodiments, targets extracted from the biological sample may befurther separated by gel electrophoresis. Separation of targets may beby isoelectric point (pI), molecular weight, electric charge, or acombination of these factors. The nature of the separation may depend onthe treatment of the sample and the nature of the gel. A suitable gelmay be selected from a polyacrylamide gel, an SDS-polyacrylamide gel, oran agarose gel.

A suitable membrane may be selected such that the membrane hasnon-specific target binding properties. In some embodiments, a suitablemembrane may be selected from a polyvinylidene fluoride membrane, anitrocellulose membrane, or a nylon membrane. In some embodiment, asuitable membrane may be selected such that the membrane may besubstantially stable to multiple probing. In embodiments involvingprobing of targets using protein probes, the membranes may be blockedusing a blocking solution to prevent non-specific binding of proteinprobes to the membranes. In embodiments, involving probing of DNAfragments, the DNA gel may be treated with a dilute HCL solution or analkaline solution to facilitate more efficient transfer of the DNA fromthe gel to the membrane.

In some embodiments, the membrane may be subjected to temperatures in arange of about 60° C. to about 100° C. to covalently bind the targets tothe membrane, for example DNA targets to a nitrocellulose membrane. Insome embodiments, the membrane may be exposed to ultraviolet radiationto covalently bind the targets to the membrane, for example DNA targetsto a nylon membrane. In some embodiments, the targets in the biologicalsample may not be separated by electrophoresis before blotting on amembrane and may be probed directly on a membrane, for example, in dotblot techniques.

Following the preparation of the tissue sample or the membrane, a probesolution (e.g., labeled-antibody solution) may be contacted with thetissue section or the membrane for a sufficient period of time and underconditions suitable for binding of binder to the target (e.g., antigen).As described earlier, two detection methods may be used: direct orindirect. In a direct detection, a signal generator-labeled primaryantibody (e.g., fluorophore-labeled primary antibody or enzyme-labeledprimary antibody) may be incubated with an antigen in the tissue sampleor the membrane, which may be visualized without further antibodyinteraction. In an indirect detection, an unconjugated primary antibodymay be incubated with an antigen and then a labeled secondary antibodymay bind to the primary antibody. Signal amplification may occur asseveral secondary antibodies may react with different epitopes on theprimary antibody. In some embodiments two or more (at most four) primaryantibodies (labeled or unlabeled) may be contacted with the tissuesample. Unlabeled antibodies may be then contacted with thecorresponding labeled secondary antibodies. In alternate embodiments, aprimary antibody and specific binding ligand-receptor pairs (such asbiotin-streptavidin) may be used. The primary antibody may be attachedto one member of the pair (for example biotin) and the other member (forexample streptavidin) may be labeled with a signal generator or anenzyme. The secondary antibody, avidin, streptavidin, or biotin may beeach independently labeled with a signal generator or an enzyme.

In embodiments where the primary antibody or the secondary antibody maybe conjugated to an enzymatic label, a fluorescent signalgenerator-coupled substrate may be added to provide visualization of theantigen. In some embodiments, the substrate and the fluorescent signalgenerator may be embodied in a single molecule and may be applied in asingle step. In other embodiments, the substrate and the fluorescentsignal generator may be distinct entities and may be applied in a singlestep or multiple steps.

An enzyme coupled to the binder may react with the substrate to catalyzea chemical reaction of the substrate to covalently bind the fluorescentsignal generator-coupled substrate the biological sample or the solidsupport to which the sample is bound. In some embodiments, an enzyme mayinclude horseradish peroxidase and the substrate may include tyramine.Reaction of the horseradish peroxidase (HRP) with the tyramine substratemay cause the tyramine substrate to covalently bind to phenolic groupspresent in the sample or a solid support to which the sample is bound.In embodiments employing enzyme-substrate conjugates, signalamplification may be attained as one enzyme may catalyze multiplesubstrate molecules. In some embodiments, methods disclosed herein maybe employed to detect low abundance targets using indirect detectionmethods (e.g., using primary-secondary antibodies), using HRP-tyramidesignal amplification methods, or combinations of both (e.g., indirectHRP-tyramide signal amplification methods). Incorporation of signalamplification techniques into the methods disclosed herein andcorrespondingly the type of signal amplification techniques incorporatedmight depend on the sensitivity required for a particular target and thenumber of steps involved in the protocol.

Observing a Signal from the Probe

A signal from the signal generator may be detected using a detectionsystem. The nature of the detection system used may depend upon thenature of the signal generators used. The detection system may includean electron spin resonance (ESR) detection system, a charge coupleddevice (CCD) detection system (e.g., for radioisotopes), a fluorescentdetection system, an electrical detection system, a photographic filmdetection system, a chemiluminescent detection system, an enzymedetection system, an atomic force microscopy (AFM) detection system (fordetection of microbeads), a scanning tunneling microscopy (STM)detection system (for detection of microbeads), an optical detectionsystem, a near field detection system, or a total internal reflection(TIR) detection system.

One or more of the aforementioned techniques may be used to observe oneor more characteristics of a signal from a signal generator (coupledwith a binder or coupled with an enzyme substrate). In some embodiments,signal intensity, signal wavelength, signal location, signal frequency,or signal shift may be determined using one or more of theaforementioned techniques. In some embodiments, one or moreaforementioned characteristics of the signal may be observed, measured,and recorded.

In some embodiments, a signal generator (coupled with a binder or couplewith an enzyme substrate) may include a fluorophore and fluorescencewavelength or fluorescent intensity may be determined using afluorescence detection system. In some embodiments, a signal may beobserved in situ, that is, a signal may be observed directly from thesignal generator associated through the binder to the target in thebiological sample. In some embodiments, a signal from the signalgenerator may be analyzed within the biological sample, obviating theneed for separate array-based detection systems.

In some embodiments, observing a signal may include capturing an imageof the biological sample. In some embodiments, a microscope connected toan imaging device may be used as a detection system, in accordance withthe methods disclosed herein. In some embodiments, a signal generator(such as, fluorophore) may be excited and the signal (such as,fluorescence signal) obtained may be observed and recorded in the formof a digital signal (for example, a digitalized image). The sameprocedure may be repeated for different signal generators (if present)that are bound in the sample using the appropriate fluorescence filters.

Applying a Chemical Agent to Modify the Signal

A chemical agent may be applied to the sample to modify the signal. Insome embodiments, signal modification may include one or more of achange in signal characteristic, for example, a decrease in intensity ofsignal, a shift in the signal peak, a change in the resonant frequency,or cleavage (removal) of the signal generator resulting in signalremoval. In some embodiments, a chemical agent may be applied to modifythe signal by substantially inactivating the fluorescent signalgenerator and the enzyme (if employed). In some embodiments, a chemicalagent may include an oxidizing agent, which may substantially oxidizethe fluorescent signal generator. In some embodiments, a chemical agentmay be applied to modify the signal by substantially oxidizing thecleavable linkage to cleave off the signal generator.

In some embodiments, a chemical agent may be in the form of a solutionand the sample may be contacted with the chemical agent solution for apredetermined amount of time. In some embodiments, a chemical agent maybe a basic solution having an oxidizing agent. The concentration of thebasic solution and the contact time may be dependent on the type ofsignal modification desired. In some embodiments, a chemical agentsolution may be contacted with the sample and the oxidation step may beperformed for less than 30 minutes. In some embodiments, the oxidationstep may be performed for less than 15 minutes. In some embodiments, theoxidation step may be performed for about 30 seconds to about 15minutes. In some embodiments, the oxidation step may be performed forabout 5 minutes. In some embodiments, the oxidation step may beperformed at room temperature.

In some embodiments, the contacting conditions for the basic solutionmay be selected such that the binder, the target, the biological sample,and binding between the binder and the target may not be affected. Insome embodiments, an oxidizing agent may only affect the signalgenerator and the enzyme (if employed) and the oxidizing agent may notaffect the target/binder binding or the binder integrity. Thus by way ofexample, a binder may include a primary antibody or a primaryantibody/secondary combination. An oxidizing agent according to themethods disclosed herein may only affect the signal generator, and theprimary antibody or primary antibody/secondary antibody combination mayessentially remain unaffected. In some embodiments, a binder (such as, aprimary antibody or primary antibody/secondary antibody combination) mayremain bound to the target in the biological sample after contacting thesample with the oxidizing agent.

In some embodiments, a binder may remain bound to the target in thebiological sample after contacting the sample with the oxidizing agentand the binder integrity may remain essentially unaffected (for example,an antibody may not substantially denature or elute in the presence of achemical agent). In some embodiments, after application of the oxidizingagent to the sample less than 25 percent of the binders may be strippedfrom the targets in the biological sample. In some embodiments, afterapplication of the oxidizing agent to the sample less than 20, less than15 percent, less than 10 percent, or less than 5 percent of the bindersmay be stripped from the targets in the biological sample.

In some embodiments, a characteristic of the signal may be observedafter contacting the sample with the oxidizing agent to determine theeffectiveness of the signal modification. For example, a color may beobserved before application of the oxidizing agent and the color may beabsent after application of the oxidizing agent. In another example,fluorescence intensity from a fluorescent signal generator may beobserved before contacting with the oxidizing agent and after contactingwith the oxidizing agent. In some embodiments, a decrease in signalintensity by a predetermined amount may be referred to as signalmodification. In some embodiments, modification of the signal may referto a decrease in the signal intensity by an amount in a range of greaterthan about 50 percent. In some embodiments, modification of the signalmay refer to a decrease in the signal intensity by an amount in a rangeof greater than about 60 percent. In some embodiments, modification ofthe signal may refer to a decrease in the signal intensity by an amountin a range of greater than about 80 percent.

Contacting the Sample with a Subsequent Probe and Binding to aSubsequent Target

The biological sample or the sample bound to a solid support may becontacted with a subsequent probe using one or more procedures describedherein above for the first probe. The subsequent probe may be capable ofbinding to target different from the target bound in the earlier steps.In embodiments where a plurality of probes may be contacted with thebiological sample in the earlier probe contact steps, the subsequentprobe may be capable of binding a target different from the targetsbound by the earlier probe set. In some embodiments, a biological samplemay be contacted with a plurality of probes in the subsequent probecontact step. In embodiments where binders coupled to enzymes may beemployed as probes, binding steps may further include reacting stepsinvolving reaction of the enzyme with an enzyme substrate coupled tofluorescent signal generator.

In some embodiments, the signal generator (e.g., a fluorescent signalgenerator) used in the different binding steps may be the same, that is,detectable in the same detection channel. Methods employing the samesignal generator in different binding steps may allow for detection ofmultiple targets when limited number of detection channels areavailable. In some embodiments, where a set of probes (2 to 4 probes)may be employed in the first binding step, the subsequent probes mayinclude the same signal generators as in the earlier binding steps. Forexample, a first binding step may include Cy3, Cy5, and Cy7-conjugateddifferent binders. In some embodiments, the subsequent binding steps mayalso include the same dye set, that is, Cy3, Cy5, and Cy7.

In some embodiments, the signal generator (e.g., a fluorescent signalgenerator) used in the different binding steps may be different, thatis, independently detectable in different detection channels. Forexample, in some embodiments, a first probe may include a Cy3 dye, whichhas a fluorescent emission wavelength in the green region and asubsequent probe may include a Cy7 dye, which has a fluorescent emissionwavelength in the red region.

In embodiments employing binder-coupled enzymes as probes, the enzymesand the substrates employed in the different binding and reacting stepsmay be the same. An oxidizing agent may inactivate the earlier enzymebefore binding the sample to a subsequent enzyme to preventcross-reaction of the earlier enzyme with the subsequent substrate. Forexample, a first binding and reacting step may include binder coupled toHRP and tyramine coupled to a first fluorophore. The oxidizing step mayinvolve the steps of substantially oxidizing the fluorophore andsubstantially inactivating the HRP. In some embodiments, oxidation andinactivation steps may occur simultaneously. In some embodiments,oxidation and inactivation steps may occur sequentially. After theoxidation and inactivation steps, the sample may be contacted with asubsequent binder coupled to HRP, which may be further reacted withtyramine coupled to a second fluorophore. Similarly, the subsequentbinding and reacting steps may be affected using multiple iterations ofHRP-tyramine as enzyme substrate conjugates, each binding and reactingstep followed by the oxidation and inactivation step. The firstfluorophore and the subsequent fluorophores may be the same or differentdepending on the number of detection channels available for detection.

In some embodiments, the first binding step may include a set of probes(e.g., 2 to 4 probes), each probe including a binder capable of bindingto a different target and each enzyme capable of catalyzing a chemicalreaction of a different substrate. For example, in one embodiment, thefirst probe set may include a binder1 coupled to HRP and a binder2coupled to AP. The reacting step may include contacting the sample withtyramine-coupled to Cy3 and NADP-coupled to Cy7. Following reaction ofthe enzymes with their corresponding substrates and observing thesignals, the cyanine dyes may be oxidized and the enzymes inactivated byaddition of a suitable oxidizing agent. The subsequent probing steps mayinclude the same set of binder-enzyme and substrate-fluorophore pairs ordifferent set of binder-enzyme and substrate-fluorophore pairs. Theplurality of probes and the substrate-signal generator may be contactedwith the biological sample simultaneously (for example, as a singlemixture) or sequentially (for example, a probe1 may be contacted withthe biological sample, followed by washing step to remove any unboundprobe1, followed by contacting a probe2 with the biological sample, andso forth).

Observing a Subsequent Signal from a Subsequent Probe.

One or more detection methods described hereinabove may be used toobserve one or more characteristics of a subsequent (e.g., second,third, etc.) signal from a subsequent signal generator (present in thesubsequent probe). In some embodiments, signal intensity, signalwavelength, signal location, signal frequency, or signal shift may bedetermined using one or more of the aforementioned techniques. Similarto the first signal, a subsequent signal (for example, a fluorescencesignal) obtained may be recorded in the form of a digital signal (forexample, a digitalized image). In some embodiments, observing asubsequent signal may also include capturing an optical image of thebiological sample.

Reiteration of the Contacting, Binding, and Observing Steps

In some embodiments, after contacting the sample with a subsequent(e.g., second, third, etc.) probe, oxidation of the signal generator,and subsequent probe administration may be repeated multiple times. Insome embodiments, after observing a second signal from the second probe,the biological sample may be contacted with an oxidizing agent to modifythe signal from the second probe. Furthermore, a third probe may becontacted with the biological sample, wherein the third probe may becapable of binding a target different from the first and the secondprobes. Likewise, a signal from the third probe may be observed followedby application of oxidizing agent to modify the signal. The binding,observing, and oxidation steps may be repeated iteratively multipletimes using an n^(th) probe capable of binding to additional targets toprovide the user with information about a variety of targets using avariety of probes and/or signal generators. In embodiments where binderscoupled to enzymes may be employed as probes, binding steps may furtherinclude reacting steps involving reaction of the enzyme with an enzymesubstrate coupled to fluorescent signal generator. In some embodiments,the oxidation, binding, reacting (if applicable), and observing stepsmay be repeated one or more time. In some embodiments, the oxidation,binding, reacting (if applicable), and observing steps may be repeatedat least 5, at least 10, or at least 20 times.

In some embodiments, a series of probes may be contacted with thebiological sample in a sequential manner to obtained a multiplexedanalysis of the biological sample. In some embodiments, a series ofprobe sets (including at most 4 probes in one set) may be contacted withthe biological sample in a sequential manner to obtained a multiplexedanalysis of the biological sample. Multiplexed analysis generally refersto analysis of multiple targets in a biological sample using the samedetection mechanism.

Contacting the Sample with One or More Morphological Stain

In some embodiments, a biological sample may include a cell or a tissue,and the sample may be contacted with a morphological stain before,during, or after the contacting step with the first probe or subsequentprobe. A morphological stain may include a dye that may stain differentcellular components, in order to facilitate identification of cell typeor disease status. In some embodiments, the morphological stain may bereadily distinguishable from the signal generators in the probes, thatis, the stain may not emit signal that may overlap with signal from theprobe. For example, for a fluorescent morphological stain, the signalfrom the morphological stain may not autofluoresce in the samewavelength as the fluorophores used in the probes.

A morphological stain may be contacted with the biological samplebefore, during, or after, any one of the aforementioned steps. In someembodiments, a morphological stain may be contacted with biologicalsample along with the first probe contact step. In some embodiments, amorphological stain may be contacted with the biological sample beforecontacting the sample with a chemical agent and after binding the firstprobe to the target. In some embodiments, a morphological stain may becontacted with a biological sample after contacting the sample with achemical agent and modifying the signal. In some embodiments, amorphological stain may be contacted with a biological sample along withthe second probe contact step. In some embodiments, a biological samplemay be contacted with the morphological stain after binding the secondprobe to the target. In some embodiments, where the morphological stainsmay result in background noise for the fluorescent signal from thesignal generator, the morphological stains may be contacted with thebiological sample after the probing, oxidizing and reprobing steps. Forexample, morphological stains like H&E may be sequentially imaged andregistered after the methods disclosed herein.

In some embodiments, chromophores, fluorophores, or enzyme/enzymesubstrates may be used as morphological stains. Suitable examples ofchromophores that may be used as morphological stains (and their targetcells, subcellular compartments, or cellular components) may include,but are not limited to, Eosin (alkaline cellular components, cytoplasm),Hematoxylin (nucleic acids), Orange G (red blood, pancreas, andpituitary cells), Light Green SF (collagen), Romanowsky-Giemsa (overallcell morphology), May-Grunwald (blood cells), Blue Counterstain(Trevigen), Ethyl Green (CAS) (amyloid), Feulgen-Naphthol Yellow S(DNA), Giemsa (differentially stains various cellular compartments),Methyl Green (amyloid), pyronin (nucleic acids), Naphthol-Yellow (redblood cells), Neutral Red (nuclei), Papanicolaou stain (a mixture ofHematoxylin, Eosin Y, Orange G and Bismarck Brown mixture (overall cellmorphology)), Red Counterstain B (Trevigen), Red Counterstain C(Trevigen), Sirius Red (amyloid), Feulgen reagent (pararosanilin) (DNA),Gallocyanin chrom-alum (DNA), Gallocyanin chrom-alum and Naphthol YellowS (DNA), Methyl Green-Pyronin Y (DNA), Thionin-Feulgen reagent (DNA),Acridine Orange (DNA), Methylene Blue (RNA and DNA), Toluidine Blue (RNAand DNA), Alcian blue (carbohydrates), Ruthenium Red (carbohydrates),Sudan Black (lipids), Sudan IV (lipids), Oil Red-O (lipids), VanGieson's trichrome stain (acid fuchsin and picric acid mixture) (musclecells), Masson trichrome stain (hematoxylin, acid fuchsin, and LightGreen mixture) (stains collagen, cytoplasm, nucleioli differently),Aldehyde Fuchsin (elastin fibers), or Weigert stain (differentiatesreticular and collagenous fibers).

Examples of suitable fluorescent morphological stains (and their targetcells, subcellular compartments, or cellular components if applicable)may include, but are not limited to: 4′,6-diamidino-2-phenylindole(DAPI) (nucleic acids), Eosin (alkaline cellular components, cytoplasm),Hoechst 33258 and Hoechst 33342 (two bisbenzimides) (nucleic acids),Propidium Iodide (nucleic acids), Spectrum Orange (nucleic acids),Spectrum Green (nucleic acids), Quinacrine (nucleic acids),Fluorescein-phalloidin (actin fibers), Chromomycin A 3 (nucleic acids),Acriflavine-Feulgen reaction (nucleic acid), Auramine O-Feulgen reaction(nucleic acids), Ethidium Bromide (nucleic acids). Nissl stains(neurons), high affinity DNA fluorophores such as POPO, BOBO, YOYO andTOTO and others, and Green Fluorescent Protein fused to DNA bindingprotein, such as histones, ACMA, Quinacrine and Acridine Orange.

Examples of suitable enzymes (and their primary cellular locations oractivities) may include, but are not limited to, ATPases (musclefibers), succinate dehydrogenases (mitochondria), cytochrome c oxidases(mitochondria), phosphorylases (mitochondria), phosphofructokinases(mitochondria), acetyl cholinesterases (nerve cells), lactases (smallintestine), acid phosphatases (lysosomes), leucine aminopeptidases(liver cells), dehydrogenases (mitochondria), myodenylate deaminases(muscle cells), NADH diaphorases (erythrocytes), and sucrases (smallintestine).

In some embodiments, a morphological stain may be stable towards anoxidizing agent, that is, the signal generating properties of themorphological stain may no be substantially affected by the oxidizingagent. In some embodiments, where a biological sample may be stainedwith a probe and a morphological stain at the same time, application ofoxidizing agent to modify the signal from the probe may not modify thesignal from the morphological stain. In some embodiments, amorphological stain may be used as a control to co-register themolecular information (obtained through the iterative probing steps) andthe morphological information (obtained through the morphologicalstains).

Contacting the Sample with One or More Control Probe

In some embodiments, a control probe may be bonded to one or moretargets in the biological sample. In some embodiments, a control probemay be bonded to the targets along with the first probe contact step. Insome embodiments, a control probe may be applied to the biologicalsample simultaneously with the first probe. In some embodiments, acontrol probe may be applied to the biological sample sequentially, thatis before or after the application of the first probe, but beforeapplication of the oxidizing agent.

A control probe may include a signal generator that is stable towards anoxidizing agent or the signal generating properties of the signalgenerator are not substantially effected when contacted with theoxidizing agent. A signal generator may include a radioisotope that isstable to the oxidizing agent or a fluorophore that is stable to theoxidizing agent. A suitable radioisotope may include P³² or H³, ¹⁴C,¹²⁵I or ¹³¹I. A suitable fluorophore may include DAPI.

In some embodiments, a suitable signal generator may be coupled to abinder to form a control probe. For example, a radioactive label may becoupled to an antibody to form a control probe and the antibody may bindto one or more target antigens present in the biological sample. Inother embodiments, a suitable signal generator may be capable of bindingto one more targets in the sample and also providing a detectablesignal, which is stable in the presence of the oxidizing agent. Forexample, a suitable control probe may be DAPI, which is capable ofbinding to nucleic acids in the sample and also capable of providing afluorescent signal that is stable to the oxidizing agent.

In some embodiments, a control probe may be employed in the methodsdisclosed herein to provide an indication of the stability of thetargets to the iterative staining steps. For example, a control probemay be bonded to a known target in the sample and a signal from thecontrol observed and quantified. The control signal may be thenmonitored during the iterative staining steps to provide an indicationof the stability of the targets or binders to the oxidizing agents. Insome embodiments, a quantitative measure (for example, signal intensity)of the control signal may be monitored to quantify the amount of targetspresent in the sample after the iterative probing steps.

In some embodiments, a control probe may be employed to obtainquantitative information of the sample of interest, for exampleconcentration of targets in the sample or molecular weight of thetargets in the sample. For example, a control target (having knownconcentration or known molecular weight) may be loaded along with thesample of interest in a blotting technique. A control probe may bebonded to the control target and a control signal observed. The controlsignal may be then correlated with the signals observed from the sampleof interest using methods described herein below.

In some embodiments, a control probe may be employed in the methodsdisclosed herein to provide for co-registration of multiple molecularinformation (obtained through the iterative probing steps) and themorphological information (obtained, e.g., using DAPI). In someembodiments, methods disclosed herein may include co-registration ofmultiple fluorescent images with the bright-field morphological imagesobtained e.g., using H&E. In some embodiments, the probes employed inthe iterative probing steps may not have any common compartmentalinformation that may be used to register with the H&E images. A controlprobe like a DAPI nuclear stain may be employed to co-register thenucleus stained with hematoxylin in the bright-field images with thefluorescent images. The fluorescent images and the bright-field imagesmay be co-registered using image registration algorithms that may begrouped in two categories: intensity-based and feature-based techniques.

Correlating the First Signal and the Subsequent Signals

In some embodiments, a first signal, a subsequent signal, or the firstsignal and the subsequent signals may be analyzed to obtain informationregarding the biological sample. For example, in some embodiments, apresence or absence of a first signal may indicate the presence orabsence of the first target (capable of binding to the first binder) inthe biological sample. Similarly, the presence or absence of a secondsignal may indicate the presence or absence of the second target(capable of binding to the second binder in the biological sample). Inembodiments where multiple targets may be analyzed using a plurality ofprobes, the presence or absence of a particular signal may indicate thepresence or absence of corresponding target in the biological sample.

In some embodiments, the observing steps may include a quantitativemeasurement of at least one target in the sample. In some embodiments,an intensity value of a signal (for example, fluorescence intensity) maybe measured and may be correlated to the amount of target in thebiological sample. A correlation between the amount of target and thesignal intensity may be determined using calibration standards. In someembodiment, intensity values of the first and second signals may bemeasured and correlated to the respective target amounts. In someembodiments, by comparing the two signal intensities, the relativeamounts of the first target and the second target (with respect to eachother or with respect to a control) may be ascertained. Similarly, wheremultiple targets may be analyzed using multiple probes, relative amountsof different targets in the biological sample may be determined bymeasuring different signal intensities. In some embodiments, one or morecontrol samples may be used as described hereinabove. By observing apresence or absence of a signal in the samples (biological sample ofinterest versus a control), information regarding the biological samplemay be obtained. For example by comparing a diseased tissue sampleversus a normal tissue sample, information regarding the targets presentin the diseased tissue sample may be obtained. Similarly by comparingsignal intensities between the samples (i.e., sample of interest and oneor more control), information regarding the expression of targets in thesample may be obtained.

In some embodiments, the observing steps include co-localizing at leasttwo targets in the sample. Methods for co-localizing targets in a sampleare described in U.S. patent application Ser. No. 11/686,649, entitled“System and Methods for Analyzing Images of Tissue Samples”, filed onMar. 15, 2007; U.S. patent application Ser. No. 11/500,028, entitled“System and Method for Co-Registering Multi-Channel Images of a TissueMicro Array”, filed on Aug. 7, 2006; U.S. patent application Ser. No.11/606,582, entitled “System and Methods for Scoring Images of a TissueMicro Array, filed on Nov. 30, 2006, and S. application Ser. No.11/680,063, entitled Automated Segmentation of Image Structures, filedon Feb. 28, 2007, each of which is herein incorporated by reference.

In some embodiments, a location of the signal in the biological samplemay be observed. In some embodiments, a localization of the signal inthe biological signal may be observed using morphological stains. Insome embodiments relative locations of two or more signals may beobserved. A location of the signal may be correlated to a location ofthe target in the biological sample, providing information regardinglocalization of different targets in the biological sample. In someembodiments, an intensity value of the signal and a location of thesignal may be correlated to obtain information regarding localization ofdifferent targets in the biological sample. For examples certain targetsmay be expressed more in the cytoplasm relative to the nucleus, or viceversa. In some embodiments, information regarding relative localizationof targets may be obtained by comparing location and intensity values oftwo or more signals.

In embodiments employing blotting techniques, the observing steps mayinclude observing a location of the signal on the blot. The location ofthe signal in the blot may be then correlated with calibration standardsloaded along with the sample in the gel to obtain information regardingthe molecular weight of the targets in the different bands. In someembodiments, a location of the signal on the blot may be correlated to amolecular weight of the target and the isoelectric point of the target,e.g., in 2D-PAGE. In some embodiments, structural proteins such as actinor tubulin may be probed using control probes in western blots toquantify the amount of targets in the sample.

In some embodiments, one or more of the observing or correlating stepmay be performed using computer-aided means. In embodiments where thesignal(s) from the signal generator may be stored in the form of digitalimage(s), computer-aided analysis of the image(s) may be conducted. Insome embodiments, images (e.g., signals from the probe(s) andmorphological stains) may be overlaid using computer-aidedsuperimposition to obtain complete information of the biological sample,for example topological and correlation information.

In some embodiments, one or more of the aforementioned may be automatedand may be performed using automated systems. In some embodiments, allthe steps may be performed using automated systems.

The methods disclosed herein may find applications in analytic,diagnostic, and therapeutic applications in biology and in medicine. Insome embodiments, the methods disclosed herein may find applications inhistochemistry, particularly, immunohistochemistry. Analysis of cell ortissue samples from a patient, according to the methods describedherein, may be employed diagnostically (e.g., to identify patients whohave a particular disease, have been exposed to a particular toxin orare responding well to a particular therapeutic or organ transplant) andprognostically (e.g., to identify patients who are likely to develop aparticular disease, respond well to a particular therapeutic or beaccepting of a particular organ transplant). The methods disclosedherein, may facilitate accurate and reliable analysis of a plurality(e.g., potentially infinite number) of targets (e.g., disease markers)from the same biological sample.

Example 1 Selective Oxidation Using Hydrogen Peroxide of Cyanine Dyeswithout Affecting DAPI

A solution of hydrogen peroxide (H₂O₂) was prepared in a sodiumbicarbonate buffer by mixing 1 volume of 1M sodium bicarbonate, 3volumes of water, and 1 volume of 30 percent (v/v) hydrogen peroxide. pHof sodium bicarbonate was adjusted to a pH 10 using sodium hydroxide,prior to mixing with hydrogen peroxide.

Three separate solutions of cyanine dyes: Cy3, Cy5, and Cy7 wereprepared in water at a concentration of about 2 μM. An aliquot of acyanine dye solution was mixed with an aliquot of the H₂O₂ solution toprepare a sample solution with a final concentration of about 3 volumepercent H₂O₂ and 1 micromolar 1 μM cyanine dye (Samples 1 (Cy3), 2(Cy5), and 3 (Cy7)). A 1 μM cyanine dye solution in water (without H₂O₂)was used as a control.

Oxidation reaction of the cyanine dye was monitored by measuringabsorbance spectrum of the dye on an ultraviolet/visible (UV/Vis)spectrophotometer as a function of time. FIG. 1 shows the absorbancespectra of Sample 1 as a function of wavelength, after duration of 10minutes and 15 minutes. The absorbance value decreased considerably whencompared with the control. FIG. 2 shows the absorbance values forSamples 1, 2, and 3 as a function of wavelength. The absorbance of theSamples 1, 2 and 3 reduced to zero after a duration of time exhibitingchemical destruction of the dye by H₂O₂. The time duration for theSamples 1, 2 and 3 was different for the different dye: 19 minutes forSample 1, 15 minutes for Sample 2, and 3 minutes for Sample 3.

A solution of 4′,6-diamidino-2-phenylindole (DAPI) was prepared in waterat a concentration of about 57 μM. An aliquot of DAPI solution was mixedwith an aliquot of the H₂O₂ solution to prepare a solution with a finalconcentration of about 3 volume percent H₂O₂ and 10 μg/mL DAPI (Sample4). A 10 μg/mL DAPI solution in water (without H₂O₂) was used as acontrol.

Oxidation reaction of DAPI was monitored by measuring absorbancespectrum of Sample 4 as a function of time. FIG. 3 shows the absorbancespectra of Sample 4 as a function of wavelength, after duration of 30minutes and 140 minutes. The absorbance value of Sample 4 did not varymuch even after a period of 140 minutes and was in the same range as thecontrol, exhibiting no significant effect of H₂O₂ on DAPI.

Example 2 Selective Oxidation (Using Sodium Periodate) of Cyanine Dyeswithout Affecting DAPI

A solution of sodium periodate (NaIO₄) was prepared by mixing a 0.2 Msolution of NaIO₄ in 0.1× phosphate buffer saline (PBS). Three separatesolutions of cyanine dyes (Cy3, Cy5, and Cy7) were prepared in water ata concentration of about 2 μM. An aliquot of a cyanine dye solution wasmixed with an aliquot of the NaIO₄ solution to prepare a solution with afinal concentration of about 1 μM NaIO₄ and 1 μM cyanine dye (Samples 5(Cy3), 6 (Cy5), and 7 (Cy7)). A 1 μM cyanine dye solution in water(without NaIO₄) was used as a control.

Oxidation reaction of the cyanine dye was monitored by measuringabsorbance spectrum of the dye on an ultraviolet/visible (UV/Vis)spectrophotometer as a function of time. FIG. 4 shows the absorbancespectra of Sample 5 as a function of wavelength, after duration of 20minutes, 60 minutes and 210 minutes. The absorbance value decreased asfunction of time when compared with the control. Complete absorbanceloss was observed after 210 minutes for Sample 5. FIG. 5 shows theabsorbance spectra of Sample 6 as a function of wavelength, afterduration of 12 minutes and 16 minutes. The absorbance value decreased asfunction of time when compared with the control. Complete absorbanceloss was observed rapidly, after 16 minutes.

A solution of 4′,6-diamidino-2-phenylindole (DAPI) was prepared in waterat a concentration of about 57 μM. An aliquot of DAPI solution was mixedwith an aliquot of the NaIO₄ solution to prepare a solution with a finalconcentration of about 0.1 M NaIO₄ and 10 μg/mL DAPI (Sample 8). A 10μg/mL DAPI solution in water (without NaIO₄) was used as a control.

Oxidation reaction of DAPI was monitored by measuring absorbancespectrum of Sample 8 as a function of time. FIG. 6 shows the absorbancespectra of Sample 8 as a function of wavelength, after duration of 22minutes, 70 minutes and 210 minutes. The absorbance value of Sample 8did not vary much even after a period of 210 minutes and a significantamount of DAPI remained intact when compared to the control.

Example 3 Selective Destruction (Using Sodium Hydroxide Base) of CyanineDyes Without Affecting DAPI

A NaOH solution was prepared at a concentration of 0.1M and 1M. Threeseparate solutions of cyanine dyes, Cy3, Cy5, and Cy7 were prepared inwater at a concentration of 2 μM. An aliquot of a cyanine dye solutionwas mixed with an aliquot of the NaOH solution of two differentconcentrations of NaOH: 0.1M NaOH (Samples 9a, 10a, and 11a) and 1M NaOH(Samples 9b, 10b, and 11b). A 1 μM cyanine dye solution in water(without NaOH) was used as a control.

Base destruction of the dye was monitored by measuring absorbancespectrum of the samples as a function of time. FIG. 7 shows theabsorbance spectra of Samples 9a, 10a, and 11a as a function ofwavelength, after duration of less than 5 minutes. The absorbance valueof Sample 9A did not vary much and a significant amount of Cy3 remainedintact when compared to the control. Sample 10a showed a 20 percentdecomposition of the Cy5 dye, while Sample 11A showed a 70 percentdecomposition of the Cy7 dye using 0.1 M NaOH. FIG. 8 shows theabsorbance spectra of Samples 9B and 10B as a function of wavelength,after duration of less than 5 minutes. Sample 9B showed a 50 percentdecomposition of the Cy3 dye, while Sample 10B showed an 80 percentdecomposition of the Cy5 dye using 1 M NaOH.

A solution of 4′,6-diamidino-2-phenylindole (DAPI) was prepared in waterat a concentration of about 57 μM. An aliquot of DAPI solution was mixedwith an aliquot of the NaOH solution of two different concentrations0.1M NaOH (Samples 12A) and 1M NaOH (Samples 12B). A 10 μg/mL DAPI dyesolution in water (without NaOH) was used as a control. Base destructionof DAPI was monitored by measuring absorbance spectrum of Samples 12Aand 12B as a function of time. FIG. 9 shows the absorbance spectra ofSamples 12A and 12B as a function of wavelength. The absorbance value ofthe samples did not vary much and a significant amount of DAPI remainedintact when compared to the control.

Example 4 Selective Destruction of Cy5 and Cy7 Dyes without AffectingCy3

A 5 percent (w/v) solution of a nucleophile was prepared by mixingtris[2-carboxyethyl]-phosphine hydrochloride (TCEP.HCl) in 1M-sodiumbicarbonate buffer (final pH 7.7). Three separate solutions of cyaninedyes, Cy3, Cy5, and Cy7 were prepared in water at a concentration ofabout 2 μM. An aliquot of a cyanine dye solution was mixed with analiquot of the TCEP.HCl solution to prepare Samples 13 (Cy3), 14 (Cy5),and 15 (Cy7). A 1 μM cyanine dye solution in water (without TCEP.HCl)was used as a control.

Destruction of the dye was monitored by measuring absorbance spectrum ofthe samples as a function of time. FIG. 10 shows the absorbance spectraof Samples 13, 14, and 15 as a function of wavelength. The absorbancevalue of Sample 13 did not vary much and a significant amount of Cy3remained intact when compared to the control after duration of 60minutes. Samples 14 and 15 showed a significant decomposition of the Cy5and Cy7 dyes after duration of 0.5 minutes.

Example 5 Selective Destruction of Cy7 Dye without Affecting Cy3

A solution of hydrogen peroxide (H₂O₂) was prepared in a phosphatebuffer saline solution (PBS) by mixing a 6% (v/v) solution of H₂O₂ in0.8×PBS (final pH 6.6). Two separate solutions of cyanine dyes, Cy3 andCy7 were prepared in water at a concentration of about 2 μM. An aliquotof a cyanine dye solution was mixed with an aliquot of the H₂O₂ solutionto prepare Samples 16 (Cy3) and 17 (Cy7)). A 1 μM cyanine dye solutionin water (without H₂O₂) was used as a control.

Destruction of the dye was monitored by measuring absorbance spectrum ofthe samples as a function of time. FIG. 11 shows the absorbance spectraof Samples 16 and 17 as a function of wavelength. The absorbance valueof Sample 16 did not vary much and a significant amount of Cy3 remainedintact when compared to the control after duration of 60 minutes. Sample17 showed a significant decomposition of the Cy7 dye after duration of60 minutes.

The following examples 6-21 illustrate embodiments of the inventionaccording to which multiple imaging of tissue samples is conducted.Multiple staining is obtained by staining, imaging, chemicallydestroying the fluorophore, restaining, imaging, and repeating the steps

Example 6 Preparation of Tissue Samples for Staining

Adult human tissue samples were obtained as tissue slides embedded inparaffin. The tissue samples included slides of colon (Biochain,T2234090), normal breast tissue (Biochain, T2234086), prostate cancer(Biochain, T2235201-1), colon adenocarcinoma (Biochain, T2235090-1),breast tissue microarray (Imgenex, IMH 367, p61), breast TMA (ImegenexIMH 367, p32), and normal prostrate (Biochain, T2234201). Paraffinembedded slides of adult human tissue were subjected to animmunohistochemistry protocol to prepare them for staining. The protocolincluded deparaffinization, rehydration, incubation, and wash.Deparaffinization was carried by washing the slides with Histochoice (ortoluene) for a period of 10 minutes and with frequent agitation. Afterdeparaffinization, the tissue sample was rehydrated by washing the slidewith ethanol solution. Washing was carried out with three differentsolutions of ethanol with decreasing concentrations. The concentrationsof ethanol used were 90 volume %, 70 volume %, and 50 volume %. Theslide was then washed with a phosphate buffer saline (PBS, pH 7.4).Membrane permeabilization of the tissue was carried out by washing theslide with 0.1 weight percent solution of Triton TX-100. Citrate bufferpH 6.0 (Vector Unmasking Solution) was used for antigen retrieval. Theslides were exposed to the buffer in a pressure cooker for a period of15 minutes followed by cooling at room temperature for 20 minutes. Theslide was then blocked against nonspecific binding by washing with PBSand 900 μL of 3 volume percent bovine serum albumin (BSA) for 45 minutesat 37 C. For staining with secondary antibodies (optional), the slidewas also blocked with 100 μL of serum from secondary antibody hostspecies.

Example 7 Conjugation of Antibodies with a Dye

Dye-conjugated antibodies were prepared according to the followingprocedure. The antibodies used for conjugating and staining includedanti-proliferating cell nuclear antigen, clone pc10 (Sigma Aldrich,P8825); anti-smooth muscle alpha actin (SmA), clone 1A4 (Sigma, A2547);rabbit anti-beta catenin (Sigma, C 2206); mouse anti-pan cytokeratin,clone PCK-26 (Sigma, C1801); mouse anti-estrogen receptor alpha, clone1D5 (DAKO, M 7047); beta catenin antibody, clone 15B8 (Sigma, C 7738);goat anti-vimentin (Sigma, V4630); cycle androgen receptor clone AR441(DAKO, M3562); Von Willebrand Factor VII, keratin 5, keratin 8/18,e-cadherin, Her2/neu, Estrogen receptor, p53, progesterone receptor,beta catenin; donkey anti-mouse (Jackson Immunoresearch, 715-166-150);and donkey anti-rabbit (Jackson Immunoresearch, 711-166-152).

A micron YM-10 spin column was wetted with 250 mL of PBS and the columnwas spun for 15 minutes. 500 mL of the antibody (200 μg/mL) was pipettedinto the wet column. The column was spun for 30 minutes at 11000 rpm at4 C. The concentrated antibody/protein was then transferred into a newtube and spun for 30 seconds to remove the concentrated protein. Acoupling buffer solution was then mixed with the concentrated antibodysolution. The coupling buffer solution included 1M sodium carbonate (pHbetween 8-9) and 5 μL of the buffer was used per 100 μL of the antibodysolution. The antibody and buffer solution was added to 0.01-0.1milligrams of the cyanine dye. The dye was reconstituted in DMSO to a10-20-mg/mL concentration prior to incubating with the antibody. Theresulting solution was mixed thoroughly by pipetting and any bubblesformed were removed by spinning the tube. The solution was covered witha foil and incubated at room temperature for a period of about 30-45minutes. Post incubation the solution was added to YM-10 spin column andspun for 30 minutes at 4 C at 11000 rpm. The solution was washed withPBS and spun to remove any unconjugated dye or antibody. Thedye-conjugated antibody solution was then diluted with 50 percentglycerol and stored in a freezer at −20 C.

Example 8 Staining and Imaging of Tissue with Dyes

A slide prepared in Example 6 was incubated with a dye-conjugatedantibody prepared in Example 7. Incubation was conducted in 3 percentBSA for 45 minutes at 37 C. After incubation, the slide was subjected toan extensive series of PBS washes. When secondary antibodies were used,the slide was incubated with a secondary antibody in BSA for 45 minutesat 37 C. After incubation, the slide was subjected to an extensiveseries of PBS washes. A primary antibody or secondary antibody-stainedslide was counterstained with the morphological stain, DAPI, and coverslipped.

A cover slipped slide was imaged using a camera. The camera used was amonochromatic Leica DFC 350FX monochromatic high-resolution cameramounted in a Leica DMRA2 fluorescent microscope. The magnification usedwas 20× unless otherwise stated. After image acquisition, the cover slipwas removed and the slide was washed with PBS to prepare for signaldestruction.

Example 9 Dye Destruction, Staining, and Imaging

NaOH solution and H₂O₂ solution were used for signal destruction. A NaOHsolution was prepared using 500 μL of 50 volume percent NaOH and 49.5 mLof PBS. The final pH of the NaOH solution was around 11.9-12.5. A H₂O₂solution was prepared by mixing 10 mL of 0.5M sodium carbonate (pH 10),5 mL of 30 volume percent H₂O₂, and 35 mL of water. A slide was placedin the NaOH or H₂O₂ solution for 15 minutes with gentle agitation. After15 minutes, the slide was washed again with PBS, cover slipped andeither imaged again (optional) to check the efficacy of the dyedestruction or restained and imaged. Restaining and reimaging steps werecarried out using the process described in Example 8. Following imaging,a slide was subjected to signal destruction, staining, and imagingcycles, and the process was repeated a multiple number of times. Thetissue samples were imaged using 1-9 different antibodies. After imagingwith the cyanine series, the slide was optionally stained and imagedwith morphological stains H&E.

Example 10 Single Channel Staining and Imaging of a Normal Colon TissueFollowed by Signal Destruction Using NaOH

A normal colon slide was stained with a primary antibody mouseanti-proliferating cell nuclear antigen (PCNA) clone pc 10, and detectedwith a Cy3-conjugated donkey anti-mouse to form Sample 18A. Sample 18Awas imaged and then treated with a NaOH solution to form Sample 18B,which was imaged again. Staining, imaging, and dye destruction stepswere performed according to the procedures described herein in Examples8 and 9. FIG. 12 shows micrographs (at 10× magnification) of Sample 18A(before dye destruction) and Sample 18B (after dye destruction). Aftertreatment with NaOH little or no signal from Cy3 remained.

Example 11 Single Channel Staining and Imaging of a Normal Colon TissueFollowed by Signal Destruction Using NaOH

A normal colon slide was stained with a primary antibody mouseanti-smooth muscle alpha actin (SmA) clone 1A4, and detected with aCy3-conjugated donkey anti-mouse to form Sample 19A. Sample 19A wasimaged and then treated with a NaOH solution to form Sample 19B, whichwas imaged again. Staining, imaging, and dye destruction steps wereperformed according to the procedures described herein in Examples 8 and9. FIG. 13 shows micrographs (at 10× magnification) of Sample 19A(before dye destruction) and Sample 19b (after dye destruction). Aftertreatment with NaOH a little amount of signal from Cy3 remained.

Example 12 Two Channel Staining and Imaging of a Normal Breast TissueUsing NaOH

A normal breast tissue was stained with a primary antibody SmA, detectedwith a Cy3-conjugated donkey anti-mouse, and counter-stained with DAPIto form Sample 20A. Sample 20a was imaged and then treated with a NaOHsolution to form Sample 20B, which was imaged again. Staining, imaging,and dye destruction steps were performed according to the proceduresdescribed herein in Examples 8 and 9. Sample 20B was restained with aprimary antibody rabbit anti-beta catenin, and detected with aCy3-conjugated anti-rabbit to form Sample 20C. Sample 20C was imaged andthen counter-stained with H&E to form Sample 20D and imaged again.

FIG. 14 shows micrographs of Sample 20A (before dye destruction) andSample 20B (after dye destruction). After treatment with NaOH little orno signal from Cy3 remained and only DAPI was observed. Micrograph ofSample 20c showed imaging in the same Cy3 channel was possible bystaining with a different antibody. Morphological information about thetissue was obtained by further staining with H&E (Sample 20D).

Example 13 Two Channel Staining and Imaging of a Prostrate Cancer TissueUsing NaOH

A prostrate cancer tissue was stained with a primary antibody mouseanti-pan cytokeratin clone PCK-26, and detected with a Cy3-conjugateddonkey anti-mouse, to form Sample 21A. Sample 21A was imaged and thencounterstained with DAPI to form Sample 21B. Sample 21B was imaged andthen treated with a NaOH solution to form Sample 21C, which was imagedagain. Staining, imaging, and dye destruction steps were performedaccording to the procedures described herein in Examples 8 and 9. Sample21C was restained with a primary antibody SmA, and detected with aCy3-conjugated anti-rabbit to form Sample 21D and imaged again.

FIG. 15 shows micrographs of Sample 21A (Cye channel) and Sample 21B(DAPI channel) before dye destruction and Sample 21C (Cye channel) afterdye destruction. After treatment with NaOH little or no signal from Cy3remained (21C) and only DAPI was observed (not shown). Micrograph ofSample 21D showed imaging in the same Cy3 channel was possible bystaining with a different antibody.

Example 14 Two Channel Staining and Imaging of a Colon AdenocarcinomaUsing NaOH

A colon adenocarcinoma slide was stained with a primary antibody mouseanti-anti-pan cytokeratin clone PCK-26, and detected with aCy3-conjugated donkey anti-mouse, to form Sample 22A. Sample 22A wasimaged and then counterstained with DAPI to form Sample 22B. Sample 22Bwas imaged and then treated with a NaOH solution to form Sample 22C,which was imaged again. Staining, imaging, and dye destruction stepswere performed according to the procedures described herein in Examples8 and 9. Sample 22C was restained with a primary antibody SmA, anddetected with a Cy3-conjugated anti-rabbit to form Sample 22d and imagedagain.

FIG. 16 shows micrographs of Samples 22A (Cye channel) and 22B (DAPIchannel) before dye destruction) and Sample 22c after dye destruction.After treatment with NaOH little or no signal from Cy3 remained (22D)and only DAPI was observed (not shown). Micrograph of Sample 22D showedimaging in the same Cy3 channel was possible by staining with adifferent antibody. Nuclear information about the tissue was obtained bystaining with DAPI (Sample 22B).

Example 15 Two Channel Staining and Imaging of a Breast TissueMicroarray with Baseline Measurement Using NaOH

A breast tissue microarray (Sample 23A) was imaged in the DAPI and Cy3channel to measure the autofluorescence from the tissue. Sample 23A wasthen stained with DAPI to form Sample 23B, imaged and then treated withNaOH to form Sample 23C, and imaged again. Sample 23A was also stainedwith a primary antibody mouse anti-estrogen receptor alpha clone 1D5,and detected with a Cy3-conjugated donkey anti-mouse, to form Sample23D. Sample 23D was imaged then treated with a NaOH solution to formSample 23E, which was imaged again. Staining, imaging, and dyedestruction steps were performed according to the procedures describedherein in Examples 8 and 9.

FIG. 17 shows micrographs of Samples 23A-E. Micrographs of Samples 23Cand 23E were compared to the autofluorescence (baseline) observed inSample 23A. Both Samples showed signal reduction. DAPI-stained sampleshowed signal reduction possibly due to destruction of nucleic acids towhich DAPI binds.

Example 16 Three Channel Staining and Imaging of Breast TMA Using NaOH

A breast sample was stained with a primary antibody mouse anti-pancytokeratin clone PCK-26, detected visualized with a Cy3-conjugateddonkey anti-mouse to form Sample 24A, and counterstained with DAPI (notshown) and imaged. Sample 24A was then treated with a NaOH solution toremove Cy3 signal (not shown), while retaining DAPI signal to formSample 24B and imaged as shown in FIG. 18 at Sample 24B Staining,imaging, and dye destruction steps were performed according to theprocedures described herein in Examples 8 and 9. Sample 24B wasrestained with a Cy3-directly conjugated beta catenin antibody to formSample 24C and imaged again. The sample was again treated with NaOH andlabeled with Cy3-direct conjugated SmA antibody to form Sample 24D andimaged again. The images obtained were registered, pseudo colored andoverlaid (Sample 24E) to give spatial information for expressingantigen. Sample 24D was further stained with H&E to form Sample 24F.

FIG. 18 shows micrographs of Sample 24A (Cy3 channel before dyedestruction) and Sample 24B (DAPI channel after dye destruction). Aftertreatment with NaOH little or no signal from Cy3 remained and only DAPIwas observed. Micrographs of Samples 24C and 24D showed imaging in thesame Cy3 channel was possible by staining with different antibodies.Morphological information about the tissue was obtained by staining withH&E (Sample 24F).

Example 17 Twelve Channel Staining and Imaging of Normal Prostrate UsingNaOH

Images were taken prior to staining to baseline the autofluorescencecoming from each channel. A normal prostrate slide was stained with acocktail of two primary antibodies: goat anti-vimentin and mouseanti-pan cytokeratin. The two primary antibodies were detected with asecond cocktail of secondary antibodies: Cy3-conjugated donkey anti-goatand Cy5-conjugated donkey anti-mouse to form Sample 25A Cy3 and Cy5respectively. Sample 25A was imaged and then treated with a NaOHsolution. The tissue was then sequentially stained with a two primaryantibodies: rabbit anti-alpha catenin, which was subsequently detectedwith Cy3 conjugated secondary antibody, and then Cy5 conjugatedanti-androgen receptor, to form Samples 25B (25b-Cy3 and 25b-Cy5respectively). Following imaging, the sample was treated with a NaOHsolution followed by staining-imaging-NaOH treatment-staining stepsusing seven Cy-directly conjugated antibodies (Samples 25C-251). Theantibodies used were: smooth muscle alpha actin, beta catenin, pancadherin, Von Willebrand Factor VII, keratin 5, keratin 8/18, ande-cadherin. Each staining step included counterstaining with DAPI(Sample 25J).

FIG. 19 shows micrographs of Sample 25A (Cy3 and Cy5 channels), Sample25B (Cy3 and Cy5 channels), and Samples 25C-25J. FIG. 19 shows thatmultiple imaging in the same Cy3 channel was possible by staining withdifferent antibodies. 12-channel multiple imaging was possible with 9 ofthe channels being Cy3 channels.

Example 18 Four Channel Staining and Imaging of Normal Prostrate UsingH₂O₂

Images were taken before staining to baseline the autofluorescencecoming from each channel. A normal prostrate slide was stained with aCy3-directly conjugated anti-pan cadherin to form Sample 26A. The slidewas imaged and treated with H₂O₂ (Sample 26B), restained withCy3-conjugated anti-vimentin (Sample 26c), treated with H₂O₂ (Sample26D), restained with Cy3-conjugated anti-pan cytokeratin (Sample 26E),treated with H₂O₂ (Sample 26F), restained with Cy3-conjugated anti-SmA(Sample 26g), and treated with H₂O₂ (Sample 26H).

FIG. 20 shows micrographs of Samples 26A-G. FIG. 20 shows that multipleimaging in the same Cy3 channel was possible by staining with differentantibodies and destroying the signal using H₂O₂.

Example 19 Residual Stain Following Staining for Abundant Proteins UsingNaOH

A normal prostrate (Sample 27A) was imaged in the Cy3 channel to measurethe autofluorescence from the tissue. Sample 27A was then stained withCy3-conjugated anti-SmA to form Sample 27B, imaged and then treated withNaOH to form Sample 27C, and imaged again. Staining, imaging, and dyedestruction steps were performed according to the procedures describedherein in Examples 8 and 9. FIG. 21 shows the micrographs of Samples27A-C. Residual stain was observed post NaOH treatment shown in sample27C.

Residual stain values were also monitored during the twelve channelsmultiple imaging described in Example 17. Using Gimp 2.2, average pixelintensities were collected for each background and NaOH treated imageand tabulated. FIG. 22 shows a plot of average pixel intensity of thebackground for each cycle in the imaging as well as a small image ofwhat the background looked like prior to staining. A large spike inresidual stain intensity was observed in cycle 4 as SmA, an abundantlyexpressed protein was stained in cycle 3.

Example 20 Residual Stain Following Staining for Abundant Proteins UsingNaOH and H₂O₂ Treatment

Two prostate slides were stained with Cy3-directly conjugated SmA(Samples 28A and 28B left panels). Both slides were given identicalpretreatment steps, concentrations, antigen retrieval and the onlydifference was signal-destruction method: method-one being with NaOH(Sample 28C), the other with H₂O₂ (Sample 28D). Two other prostateslides were stained with Cy3-directly conjugated pan cadherin (Samples29A and 29B right panels). Both slides were given identical pretreatmentsteps, concentrations, antigen retrieval and the only difference wassignal destruction method; method one being with NaOH (Sample 29C), theother with H₂O₂ (Sample 29D).

FIG. 23 shows head-to-head micrographs of SmA and pan cadherin stainingand signal removal. H₂O₂ showed more efficient dye removal for both SmAand pan cadherin when compared to NaOH.

Example 21 Residual Stain Following Multiple Cycle Staining for AbundantProteins Using NaOH and H₂O₂ Treatment

Two prostate slides were stained with Cy3-directly conjugated pancadherin (Samples 30A and 30B). Both slides were given identicalpretreatment steps including antigen retrieval and the only differencewas signal-destruction method; method one being with NaOH, the otherwith H₂O₂. The slides were subjected to 9 mockstaining-and-signal-destruction cycles before staining withCy3-conjugated pan-cytokeratin to produce Sample 30C and Sample 30D inFIG. 24.

FIG. 24 compares staining from first cycle pan cadherin stain to the9^(th) cycle of pan cytokeratin using NaOH or H₂O₂ to destroy the signalafter each staining. FIG. 25 compares background from after dye removal(Samples 30E and 30F) in the first cycle and after 9 cycles of NaOH orH₂O₂ treatment (Samples 30C and 30D). H₂O₂ (Sample 30D, FIG. 25) showedmore efficient dye removal of pan keratin when compared to NaOH (Sample30C, FIG. 25) after the 9^(th) step FIG. 26 is a plot of average pixelintensities for the background of each cycle for the NaOH or H₂O₂slides. As the plot in FIG. 26 shows, the background for the H₂O₂ slidewas significantly less for each cycle after the initial baseline.

Example 22 Antibody Stability to Chemical Agents

A colon tissue slide was stained with a primary antibody rabbitanti-beta catenin and detected with a Cy3-conjugated donkey anti-rabbitsecondary antibody to form Sample 31A. Sample 31A was imaged and thentreated with a NaOH solution to form Sample 31B, which was imaged again.Staining, imaging, and dye destruction steps were performed according tothe procedures described herein in Examples 8 and 9. Sample 31B wasrestained with a Cy3-conjugated anti-rabbit secondary antibody to formSample 31C, and imaged again. FIG. 27 shows micrographs of Samples31A-C. FIG. 27 shows that the primary antibody remains bound to thesample after NaOH treatment.

A colon tissue slide was stained with a primary antibody mouse-anti-PCNAand detected with a Cy3-conjugated donkey anti-mouse secondary antibodyto form Sample 32A. Sample 32A was imaged and then treated with a NaOHsolution to form Sample 32C, which was imaged again. Staining, imaging,and dye destruction steps were performed according to the proceduresdescribed in Examples 8 and 9. Sample 32B was restained with aCy3-conjugated anti-mouse secondary antibody to form Sample 32C, andimaged again. FIG. 28 shows that the primary antibody is still bound tothe sample after treatment with NaOH.

The following examples 23-26 illustrate embodiments of the inventionaccording to which multiple imaging of tissue samples is conducted usingenzyme-substrate-fluorophore conjugates. Multiple staining is obtainedby staining, imaging, chemically destroying the fluorophore, restaining,imaging, and repeating the steps

Example 23 Conjugation of Antibodies with an Enzyme

The antibodies used were rabbit anti-β-catenin directly conjugated tohorse radish peroxidase (HRP) enzyme, mouse anti-keratin 5 and donkeyanti-mouse conjugated to HRP antibody. The substrate for the HRP enzymewas Cy5-conjugated tyramide substrate.

Example 24 Staining and Imaging of Tissue with HRP

A slide prepared in as Example 6 was incubated with HRP-conjugatedanti-β-catenin antibody prepared in Example 23. Primary antibodyincubation was conducted in 3 percent BSA for 45 minutes at 37° C. Afterincubation, the slide was subjected to an extensive series of PBSwashes. The HRP-stained slide was incubated with Cy5-conjugated tyramidesubstrate in PBS/0.1% Triton X-100 for 10 minutes. After incubation, theslide was subjected to an extensive series of PBS washes. The Cy5tyramide substrate was then imaged with a Zeiss Axio Imager. Themagnification used was 20× unless otherwise stated. FIG. 29 shows amicrograph of Sample 33 after staining for β-catenin. After imageacquisition, the slide was washed with PBS to prepare for signaldestruction.

Example 25 Modification of Signal, Staining and Imaging

H₂O₂ solution was used for signal destruction. A H₂O₂ solution wasprepared by mixing 10 mL of 0.5 M sodium carbonate (pH 10), 5 mL of 30volume percent H₂O₂, and 35 mL of water. A slide was placed in the H₂O₂solution for 15 minutes with gentle agitation. After 15 minutes, theslide was washed again with PBS (Sample 34), coverslipped and imagedagain to check the efficacy of the dye destruction. The reimaging stepwas carried out using the process described above in Example 24. FIG. 29shows a micrograph of Sample 34 after signal modification showingsubstantial removal of signal after signal destruction. Sample 34 wasthen re-incubated with Cy5-tyramide to determine residual activity ofthe HRP enzyme and imaged as above. FIG. 29 shows a micrograph of Sample35 and no further Cy5 and HRP enzyme is chemically inactivated by H₂O₂solution such that no further enzyme-substrate reaction occurs.

Example 26 Restaining and Reimaging of Tissue with Dye

The slide (Sample 35) from Example 25 was incubated mouse anti-keratin 5antibody. Incubation was conducted in 3 percent BSA for 45 minutes at 37C. After incubation, the slide was subjected to an extensive series ofPBS washes. The keratin 5 antibody was then detected with an HRPconjugated donkey anti-mouse antibody in BSA for 45 minutes at 37° C.The HRP-stained slide was incubated with Cy5-conjugated tyramidesubstrate as in Example 25. After incubation, the slide was subjected toan extensive series of PBS washes. The Cy5-restained slide (Sample 36)was counterstained with the morphological stain, DAPI, and coverslipped.The coverslipped slide was re-imaged using the process described abovein Example 24. FIG. 29 shows a micrograph of Sample 36 after stainingfor k5 protein.

The following Examples 27-31 illustrate embodiments of the inventionaccording to which multiple imaging of dot blots is conducted. Multiplestaining is obtained by staining, imaging, chemically destroying thefluorophore, restaining, imaging, and repeating the steps

Example 27 Immobilization of Targets on a Blot

Target proteins, β-catenin peptide (Sigma 26561-1 lot R1078-005) and CEAantigen were spotted on a polyvinylidene fluoride (PVDF) membrane atthree different concentrations: 1 micrograms, 100 nanograms, and 10nanograms. The blot was blocked using 5% milk, in 1×TBS-T (Tris-bufferedsaline Tween 20 buffer) for 1 hour at room temperature.

Example 28 Staining of β-Catenin and Imaging of Blots

A blot prepared in Example 27 was incubated with a primary antibody,rabbit anti-β-catenin antibody (Sigma, C7738). Incubation was conductedat 1:200 dilution of antibody using 1 percent milk in 1×TBS-T for 1 hourat room temperature (RT). After incubation, the blot was subjected to anextensive series of washes. The blot was then incubated with adye-conjugated secondary antibody donkey anti-rabbit Cy5 (prepared as inExample 6) at 1:500 dilution using 1 percent milk in 1×TBS-T for 1 hourat room temperature (RT). After incubation, the blot (Sample 37) waswashed with 1×TBS-T. Images of the blot (Sample 37) were captured onTyphoon Imager (GE Healthcare) using Cy5 channel and a voltage settingof 450V. FIG. 30 shows an image of Sample 37. As shown in FIG. 30, spotsof variable signal intensities are observed for the three differentconcentrations of β-catenin target protein.

Example 29 Dye Destruction, and Imaging

H₂O₂ solution was used for signal destruction. A H₂O₂ solution wasprepared by mixing 10 mL of 0.5M sodium carbonate (pH 10), 5 mL of 30volume percent H₂O₂, and 35 mL of water. The blot prepared in Example 28was placed in the H₂O₂ solution for 15 minutes at room temperature withgentle agitation. After 15 minutes, the blot was washed three timesusing 1×TBS-T for 5 minutes to form Sample 38. The blot (Sample 38) wasreimaged and images of the blot were captured on Typhoon Imager (GEHealthcare) using Cy5 channel and a voltage setting of 450V. FIG. 30shows an image of Sample 38. As shown in FIG. 30, no spots were observedafter the dye destruction step indicating substantially completedestruction of Cy5.

Example 30 Staining of CEA Antigen and Imaging of Blot

The blot from Example 29 was incubated with a primary antibody mouseanti-CEA antibody. Incubation was conducted at 1:200 dilution using 1percent milk in 1×TBS-T for 1 hour at room temperature (RT). Afterincubation, the blot was subjected to an extensive series of washes. Theblot was then incubated with a dye-conjugated secondary antibody donkeyanti-mouse Cy5 (prepared in Example 6) at 1:500 dilution using 1 percentmilk in 1×TBS-T for 1 hour at room temperature (RT). After incubation,the blot (Sample 39) was subjected to an extensive series of washes.Images of the blot (Sample 39) were captured on Typhoon using Cy5channel and a voltage setting of 450V. FIG. 30 shows an image of Sample39. As shown in FIG. 30, spots of variable signal intensities areobserved for the three different concentrations of CEA antigen. TheExample illustrates that two different targets may be detected using asingle signal generator.

Example 31 Quantification of Signal after Each Step

Samples 37, 38, 39, and 40 were further analyzed to quantify the signalsin the three detected spots for each peptide (a representative exampleof regions chosen for analysis is shown in FIG. 30 with regionshighlighted by a square numbered 4-9 in FIG. 30). The signals from eachsample each of the peptide spots were normalized to an average of threebackground spots 1, 2, and 3 as shown in FIG. 30. FIG. 31 is a plot ofrelative signal intensities of the spots for Samples 37, 38, 39, and 40,showing that signal intensities reduce 10-fold after the dye-destructionstep (Sample 38).

The following examples 32-34 illustrate embodiments of the inventionaccording to which the fluorophore is destroyed by an oxidizing agentwithout significantly stripping the probe (primary antibody) from thesolid-support.

Example 32 Preparation of Cell Cultures

Alexa 488, BODIPY FL C5 (D6184), hydroxycoumarin (H1193) were obtainedfrom Invitrogen (Carlsbad, Calif.); ATTO 495, ATTO 635, and ATTO 655,were obtained from ATTO-TEC (Siegen, Germany); DY-734-NHS was obtainedfrom Dyomics (Jena, Germany); Fluorescein cadaverine was obtained fromBiotium Inc. (Hayward, Calif.). Goat anti-mouse IgG-Cy3 and goatanti-mouse IgG-Cy5 were obtained from Jackson ImmunoResearchLaboratories, Inc. (West Grove, Pa.). DPBS and PBS were obtained fromInvitrogen; 16% PFA were obtained from Electron Microscopy Sciences(Hatfield, Pa.). Goat serum was obtained from Vector Laboratories(Burlingame, Calif.). All other reagents were obtained from Sigma (St.Louis, Mo.).

LS174T cells (ATCC, CL-188) expressing carcino embryonic antigen (CEA)were cultured in EMEM (ATCC, cat#30-2003) Supplemented with 10% FBS.Cells were incubated at 37° C. and 5% CO₂. Upon 90% confluence, theywere subcultured or seeded into 96-well-plates and continued to grow foradditional two days.

Confluent cells on 96-well-plates were rinsed with DPBS (with calcium)twice, and then 2% PFA (final concentration) was added to fix cells for10 minutes, followed by permeabilization using 0.1% Triton X-100 in PBSfor 5 minutes. Next, cells were incubated in a blocking buffer (10% goatserum+3% BSA) for 1 hour at room temperature. Mouse anti-CEA (finalconcentration 10 μg/ml in 3% BSA) was added immediately after theblocking step and incubated overnight.

Example 33 Contacting the Primary Antibody with Oxidizing Agents

To investigate the effect of oxidizing agents on primary antibody,various oxidizing agent solutions (as described in Table 1) were addedto half the plate for 30 minutes, and then rinsed thoroughly with PBS.The oxidizing reagents employed were hydrogen peroxide, aqueous bromine,potassium permanganate, sodium dichromate, I₂/KI solution, t-butylperoxide, and t-butyl hydrogen peroxide. The other half of the plate wasused as control with parallel PBS incubations and washes and nooxidizing agent solutions were applied. To test the effect of a base onthe probe different bases, such as NaOH, aqueous DBU(diazobicyclos-undecene), and aqueous butyl amine were also contactedwith the plate. PBS was applied as a control agent.

All wells were incubated with 50 μl/well goat anti-mouse IgG-Cy3 or goatanti-mouse IgG-Cy5 (1:200 dilution). The whole plate was scanned usingGemini M5 spectrophotometer (Molecular Device, Sunnyvale, Calif.). Atotal of five spots were scanned in each well and then averaged to getone reading for each well. For Cy3 dye, fluorescence was read atexcitation wavelength at 544 nm, emission 580 nm, and cutoff 570 nm. ForCy5, Excitation 640 nm, Emission 675, and cutoff 665.

Table 1 shows the fluorescence values measured after application of theoxidizing agent to the primary antibody as shown in columns 2 (Cy3) and4 (Cy5). The values in the Table are normalized fluorescence intensity,that is, a ratio of the fluorescence intensity measured for wellscontacted with the oxidizing agent to the fluorescence intensitymeasured for the control wells (no oxidizing solution applied)multiplied by 100. Therefore, a value close to 100% means that theoxidizing solution has no effect on primary antibody when compared toits control (no oxidizing solution applied).

Table 1 shows that some oxidizing agent (e.g. H₂O₂) have almost noeffect on primary antibody (P>0.1), whereas in other cases, theconcentration of the oxidizing agent may be varied (e.g., for I₂/KI)such that the primary antibody is not effected substantially. The basesemployed (NaOH and butyl amine) exhibited substantial destruction of theprimary antibodies across a variety of concentrations employed. PBSshowed no effect on the primary antibody.

Example 34 Contacting the Fluorophores with Oxidizing Agents

To test the effect of oxidizing agent on fluorophore-labeled cells,various oxidizing agent solutions (as described in Table 1) were addedto different rows (control part of the plate) for 30 minutes. The platewas read again after thorough washing as described in Example 33.

Table 1 shows that some oxidizing agent (e.g. H₂O₂) have almost noeffect on primary antibody but result in substantial loss offluorescence properties for the cyanine dye. The data reported in Table1 further shows that fluorescent signal destruction worked better athigh pH for H₂O₂ solutions. As described herein, in other cases, theconcentration of the oxidizing agent may be varied (e.g. for I₂/KI) suchthat the primary antibody is not affected substantially but the cyaninedye is. The bases employed (NaOH and butyl amine) exhibited substantialloss of signal from the cyanine dye but this may be due to destruction(or stripping) of the primary antibodies by the base. PBS showed noeffect on the primary antibody and the cyanine dye.

TABLE 1 Normalized fluorescence intensity measured after contacting theplate with different oxidizing agents. On primary Bleaching BleachingCy3 (up to 6 Cy3 (up to On Primary Cy5 Bleaching Solution sets) 4 sets)Cy (2 sets) (1 set) Oxidizing reagents 3% H₂O₂, pH10 105% 11% 124%   0%3% H₂O₂ pH5  99% 73% Aqueous Bromine 100% 14% 4% potassium  0%  0%  1% 0% permanganate 1% potassium  80%  4% permanganate 4% sodium 111% 69%88% 25% dichromate.2H₂O 4% sodium 213% 10% dichromate.2H₂O in Aceticacid 5% I₂, 10% KI  9%  0%  7%  4% 0.5% I₂, 1% KI  82% 10% 0.05% I₂,0.1% KI 101% 72% 3.5% t-butyl  45% 103%  49% 84% peroxide inAcetonitrile 3.5% t-butyl 166% 65% 107%  44% hydroperoxide Bases 0.35 NNaOH  17% 11% 14% 0.1 N NaOH  37% 13% 34% 5% DBU  40% 12% 40% 1% DBU 38% 41% 5% Butylaminine  20% 13% 38% Control PBS 108% 79% 91% 96%

Example 35 Measurement of Dye-Bleaching Spectrum

Optical density (O.D.) was used to monitor dye destruction. 10 μl of dyewas mixed with equal volume of oxidizing solution and then 2 μl of themixed solution was loaded to ND-1000 Spectrophotometer (NanopropTechnologies, Inc. Wilmington, Del.). Optical density (O.D.) was read ata full spectrum ranging from 200 nm to 750 nm. For kinetic study,measurements were taken at time 0, 5 min, 15 min, and 30 min.

Example 36 Kinetic study of Cy3 and Cy5 Destruction with H₂O₂

To study the kinetics of dye destruction, hydrogen peroxide (1.5% H₂O₂at pH 10) was used as an oxidizing agent and Cy3 and Cy5 asrepresentative dyes using the method describe in Example 36. Thefluorescence spectrum of the dye-oxidizing solution mixture wasmonitored for a period of 30 minutes. FIG. 32 shows the time profile ofspectrums of Cy3 and Cy5. A rapid decrease in O.D. was observed,indicating dye destruction. In both cases, 15 minute incubation in 1.5%H₂O₂ could practically destroy the dyes. O.D. values at peak excitationreduced to 2% for both Cy3 and Cy5 after 15 minutes.

Example 37 Effect of H₂O₂ Concentrations on Cy3 Bleaching

A range of H₂O₂ concentrations from 0 to 1.5% were tested and absorbancespectrum was monitored over 30 minutes using the method described inExample 35. The pH of H₂O₂ solution was kept at 10. Cy3 was used as adye, and absorbance was recorded at its peak wavelength at 550 nm.Except for 0.1% H₂O₂, all other concentrations of H₂O₂ showed verysimilar results, and almost reduced dye absorbance to 5% within 15minutes as shown in FIG. 33. There is no statistical difference amongtreatments with concentrations of 0.5%, 1% and 1.5% (P>0.1).

Example 38 Effect of H₂O₂ on Various Fluorophores

A panel of fluorescent dyes was tested for the effect of H₂O₂ (3% H₂O₂at pH 10) on different fluorophores using the method described inExample 35. Absorbance spectrum was monitored over 30 minutes and theabsorbance values are shown in FIG. 34. Data shown here are O.D. valuesat dye's maximum absorbance wavelength normalized to O.D. at time 0.Fluorescein, which is very sensitive to photobleaching, was relativelystable to this oxidizing method. ATTO 496 and Alexa 488, were also lesssensitive to the oxidizing agent when compared to other dyes. FIG. 34shows that the dyes could be destroyed using H₂O₂.

Example 39 Effect of H₂O₂ on Quantum Dots (QD 655)

A range of H₂O₂ concentrations (from 20× to 2000× dilution) were testedand absorbance spectrum was monitored over 30 minutes using the methoddescribed in Example 36. The pH of H₂O₂ solution was kept at 10. Theabsorbance values were compared to a control sample of a solution of QD655 at different concentrations (from 20× to 2000× dilution).

Qdot 655 goat F(ab′)2 anti-mouse IgG conjugate (1 uM) solution wasdiluted 10×, 100× or 1000× with 0.775M sodium bicarbonate solution(adjusted to pH 10). To each solution an equal volume of 6% hydrogenperoxide was added. 100 ul aliquots of each in triplicates were placedin a 96 well microtitre plate. Samples without peroxide but diluted tothe same final concentration with water were used as controls. A 2×diluted bicarbonate solution was used as background control.Fluorescence was measured over time on spectromax M2 plate reader usingfollowing parameters: Excitation 350 nm, Emission 655 nm and auto cutoff 630 nm. For the lowest concentration, 99% reduction in fluorescencewas achieved after 5 min. With in 30 min, fluorescence of all peroxidesamples was reduced by >99.5%. Fluorescence of control samples wasmostly intact (>93% for the lowest conc. sample and ˜99% for the highestconc. sample).

Three percent hydrogen peroxide in bicarbonate buffer (pH 10) was usedto destroy signal. The absorbance values were compared to a controlsample of a solution of QD 655 at different concentrations (from 20× to2000× dilution). At all concentrations of qdot the fluorescence reducedto less than 6% of the control value within 5 minutes as shown in FIG.35.

Example 40 Effect of Hydroxyl Radicals on Fluorescein

Absorbance spectra of the fluorescein dye FAM cadaverine (“FAM-CAD”)were measured using different oxidizing agent conditions for Samples 41(FAM-CAD, H₂O₂ and H₂O (1:1:1), 42 (FAM-CAD, H₂O₂ and FeCl₃ (1:1:1)), 43(FAM-CAD, H₂O and FeCl₃ (1:1:1)), 44 (FAM-CAD and H₂O (1:2)). A solutionof Fluorescein-cadaverine was mixed with 9% hydrogen peroxide and a 2%solution of FeCl₃ in water in the ratio of 1:1:1 by volume. Mixture wasallowed to stand for 5 minutes. Since the addition of FeCl₃ changes pHto highly acidic and fluorescein spectrum is known to be different underacidic conditions, prior to UV/VIS spectrum measurement, solution wasmade basic by addition of 1N NaOH, filtered and its spectrum wasrecorded. Solutions similarly treated except where either the hydrogenperoxide was replaced with water or FeCl₃ solution was replaced withwater were used as controls. A solution where both FeCl₃ and hydrogenperoxide were replaced with water was also used as another control.Although in the presence of peroxide, spectra are a bit noisy andbaseline is raised due to bubble formation, spectra clearly showed thatwhile control samples were unaffected, practically all of thefluorescein was destroyed in the test sample. The absorbance reducedconsiderably after 5 minutes for Sample 42 as shown in FIG. 36,indicating that fluorescein can be destroyed by employing an oxidizingagent to create hydroxyl radicals.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects asillustrative rather than limiting on the invention described herein. Thescope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes that come within themeaning and range of equivalency of the claims are therefore intended tobe embraced therein. The invention may be embodied in other specificforms without departing from the spirit or essential characteristicsthereof. The foregoing embodiments are therefore to be considered in allrespects as illustrative rather than limiting on the invention describedherein. The scope of the invention is thus indicated by the appendedclaims rather than by the foregoing description, and all changes thatcome within the meaning and range of equivalency of the claims aretherefore intended to be embraced therein.

1. A method of probing multiple targets in a biological samplecomprising: (a) providing a biological sample containing multipletargets adhered to a solid support; (b) binding at least one fluorescentprobe to one or more targets present in the sample; (c) observing asignal from the fluorescent probe bound in step (b); (d) oxidizing thebound fluorescent probe with a solution comprising an oxidizing agentthat substantially inactivates the fluorescent probe; (e) binding atleast one fluorescent probe to one or more targets present in the sampleof step (d); and (f) observing a signal from the fluorescent probe boundin step (e).
 2. The method of claim 1, wherein the solution in step (d)is a basic solution.
 3. The method of claim 2, wherein the basicsolution has a pH of about
 10. 4. The method of claim 2, wherein thebasic solution does not contain a reducing agent or a surfactant.
 5. Themethod of claim 1, wherein the oxidation step is performed withoutstripping more than, or about, 20% of the fluorescent probe from thetarget adhered to the solid support.
 6. The method of claim 1, whereinthe oxidizing agent is selected from hydrogen peroxide, potassiumpermanganate, sodium dichromate, aqueous bromine, iodine-potassiumiodide, and t-butyl hydroperoxide.
 7. The method of claim 1, wherein thefluorescent probe comprises a binder and a fluorescent signal generator.8. The method of claim 7, wherein the fluorescent signal generatorcomprises a cyanine dye.
 9. The method of claim 1, wherein the samplecomprises whole cells or tissue sections.
 10. The method of claim 1,wherein the sample comprises proteins or nucleic acids.
 11. The methodof claim 1, wherein steps (d)-(f) are repeated one or more times. 12.The method of claim 1, wherein steps (d)-(f) are repeated at least 5, atleast 10, or at least 20 times.
 13. The method of claim 1, wherein theoxidation step (d) is performed for less than, or about, 30 minutes. 14.The method of claim 1, wherein the oxidation step (d) is performed forabout 30 seconds to about 15 minutes.
 15. The method of claim 1, whereinthe oxidation step (d) is performed at room temperature.
 16. The methodof claim 1, further comprising measuring one or more intensity values ofthe signal observed in observing step (c), step (f), or steps (c) and(f).
 17. The method of claim 16, further comprising correlating theintensity value with an amount of target present in the sample.
 18. Themethod of claim 7, wherein the fluorescent signal generator in step (b)is the same as the fluorescent signal generator in step (e).
 19. Themethod of claim 7, wherein the fluorescent signal generator in step (b)is different from the fluorescent signal generator in step (e).
 20. Themethod of claim 1, wherein the signals observed in step (c) and step (f)are both detectable in a single detection channel.
 21. The method ofclaim 1, wherein the signal observed in step (c) or step (f) isindependently detectable in different detection channels.
 22. A methodof probing multiple targets in a biological sample comprising: (a)providing a biological sample containing multiple targets adhered to amembrane; (b) binding at least one fluorescent probe to one or moretargets present in the sample; (c) observing a signal from thefluorescent probe bound in step (b); (d) oxidizing the bound fluorescentprobe with a solution comprising an oxidizing agent that substantiallyinactivates the fluorescent probe; (e) binding at least one fluorescentprobe to one or more targets present in the sample of step (d); (f)observing a signal from the fluorescent probe bound in step (e).
 23. Themethod of claim 22, wherein the membrane is selected from nylon,agarose, nitrocellulose, and polyvinylidene difluoride.
 24. The methodof claim 22, further comprising separating the multiple targets byelectrophoresis before step (a).